Method for identifying neurite-growth promoting agents

ABSTRACT

The present invention provides methods of identifying and using compounds that modulate cell motility. Such compounds can be used to inhibit cancer cell metastasis or promote neurite growth and regenaration. The methods generally relate to the repellent-receptor signalling pathway that controls cellular attachment and detachment to a substratum.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. §371 ofPCT Application No. PCT/US99/11320, filed May 21, 1999, which claims thebenefit of priority under 35 U.S.C. §119(e) to U.S. ProvisionalApplication Serial No. 60/085,477, filed May 22, 1998.

GOVERNMENT RIGHTS

This invention was supported in part with funding provided by NIH GrantNo. NS24672, awarded by the National Institutes of Health, and by BNSGrant No. 9109775, awarded by the National Science Foundation. Thegovernment may have certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to methods for identifying and usingcompounds that inhibit cancer cell metastasis or promote neurite growthand regeneration.

BACKGROUND OF THE INVENTION

Amoeboid locomotion requires a quasi-metastable state of the adhesionsites, i.e., of the interactions between (i) the cell's contractileapparatus, (ii) adhesion molecules and other plasma membrane components,and (iii) the growth substratum. FIG. 1 plots in principle therelationship between the strength of cell adhesion to the substratum andcell motility. At the extreme ends of the curve, locomotion is reducedto zero. At these ends, the cell is either too tightly adherent, or itis so loosely attached that force generation against the substratum isimpossible. Optimum motility requires an intermediate, dynamic statethat facilitates the making and breaking of adhesions as the cell moves.In other words, a factor that increases motility would not be expectedto increase or decrease attachment but to facilitate assembly anddisassembly of adhesion sites. Knowledge of the attachment mechanism hasincreased significantly in recent years, but knowledge about detachmenthas been rudimentary until recently. Both processes must be elucidatedto understand the motility of metastatic cancer cells, and the influenceof motility factors.

For example, chemorepellents provide important guidance cues for growthcones during nervous system development. These repellents causedeveloping neurites to change their course of outgrowth away from therepellent source and, thus, play a critical role in pathfinding forgrowing or regenerating nerves. However, the mechanisms of action of therepellents are not well understood.

The establishment of metastases is a complex, multi-step phenomenon thatbegins with dissociation of cancer cells from the primary tumor andinvasion of surrounding tissues. Although understanding of themetastatic process is incomplete, at least four elements (in addition tocontinued cell proliferation) have been identified in recent years: (i)changes in cell adhesion molecules, (ii) secretion/surface expression ofproteases, (iii) increased cell motility, and (iv) vascularization ofprimary and secondary tumors. In addition, factors that promotemetastatic progression include genetic instability and defects incell-cell signaling. For example, loss of the NDP kinase-like proteinencoded by nm23, a putative metastasis-suppressor gene, seems to resultin altered signaling responsiveness, e.g., in motility assays describedin MacDonald et al., J. Biol. Chem. 268:25780-25789 (1993) and Kantor etal., Cancer Res. 53:1971-1973 (1993). Motility has been correlated withmetastatic potential as reported in Guirguis et al., Nature, 329:261-263(1987); Partin et al., Cancer Res, 48:6050-6053 (1988); Partin et al.,Proc Natl Acad Sci. USA, 86:1254-1258 (1989); Mohler, supra.; andStearns & Steams, Cancer Metastasis Rev. 12:39-52 (1993).

A growing number of factors contributing to metastastic progression arebeing identified. The recently identified KAI 1 gene encodes a putativecell adhesion molecule whose expression reduces prostate carcinoma cellmotility and metastasis. However, a universal prognostic marker ofprostate carcinoma has not been identified to date. Therefore, athorough understanding of the mechanisms that trigger invasive cancercell behavior is particularly important for prostate carcinoma.

Vertebrate amoeboid cell systems, such as polymorphonuclear leukocytes,platelets and the nerve growth cone (the pseudopodal, enlarged leadingedge of the growing nerve fiber) have been studied in some detail.Pseudopods of locomoting cells are filled with actin microfilaments, andthere is considerable knowledge of the components involved in theregulation of polymerization and of force generation in the actin-basedcytoskeleton. At so-called focal adhesion sites, the cytoskeletoninteracts with the plasma membrane and, via adhesion molecules, with theextracellular matrix or adhesion molecules on neighboring cells. To makelocomotion possible, attachment of adhesion molecules to the growthsubstratum has to be regulated coordinately with the binding of theseadhesion molecules, via linker proteins, to the actin cytoskeleton.Numerous proteins are involved in the intracellular interactions. Theyinclude, among others, talin, vinculin, Src family non-receptor tyrosinekinases, focal adhesion kinase, certain types of protein kinase C, andthe protein kinase substrate, myristoylated alanine-rich C-kinasesubstrate (MARCKS) (Burridge et al., Ann Rev Cell Biol. 4:487-525(1988); Jaken et al., J. Cell Biol., 109:697-704 (1989); Luna and Hitt,Science 258:955-964 (1992); Blackshear, J. Biol. Chem., 268:1501-1504(1993); Schaller and Parsons, Trends Cell Biol. 3:258-262 (1993)).

There are several classes of adhesion molecules. At least in the case ofintegrins and cadherins, there is evidence that they function not onlyas adhesion molecules, but also as receptors that signal ligand bindingacross the membrane. Conversely, external ligand affinity can bemodulated by integrin or cadherin phosphorylation on the inside of thecell. Outside-in signaling triggers focal adhesion assembly by a processknown to require tyrosine kinase activity. In summary, adhesion sitesare distinctive cell organelles comprised of protein assemblies thatregulate cell attachment and play an important role in amoeboidmotility.

A variety of amoeboid systems, including macrophages, platelets andnerve growth cones, exhibit high activity of cytosolic phospholipase A₂(cPLA₂) and generate high levels of cytosolic arachidonic acid (AA). Inplatelets, cPLA₂ is activated via the thrombin receptor. Also,ras-transfected cancer cells with increased motility exhibit increasedcPLA₂ activity. This suggests a role for cPLA₂ and its product, AA, inthe regulation of cell motility. The eicosanoid,12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) has long been known toaffect leukocyte motility and has been implicated in cancer cellattachment. 12-lipoxygenase (12-LOX), is the enzyme that converts AAinto 12-hydroperoxyeicosatetraenoic acid, which is reduced spontaneouslyto 12(S)-HETE. A correlation between metastatic potential and expressionlevels of 12-LOX has been reported in Honn et al., Cancer MetastasisRev. 13:365-396 (1994). These observations also implicate AA and HETEsin the regulation of cell attachment and/or motility.

There is considerable interest in eicosanoids as they relate to theprostate because unsaturated fatty acids inhibit steroid 5α reductaseand lowered AA levels have been correlated with increased malignancy ofprostate carcinoma cells. In addition, 12-LOX is elevated inadvanced-stage human prostate carcinoma.

Results obtained by other laboratories and discussed above weregenerated in isolation and never synthesized in the manner describedherein. Furthermore, the signaling pathway mediating cellular shape andmotility responses to thrombin had not been elucidated. In fact, priorto the present invention, functional assays were performed in vivo or inculture with intact cells and monitored a combination of cellularbehaviors such as cell adhesion, detachment and motile behavior.

Accordingly, a need exists for assays that can quickly and selectivelyidentify agents that modulate cell adhesion and detachment. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention generally relates to methods of identifying agentsthat modulate the motility of cells. In one aspect, the methods areaccomplished by (a) attaching pseudopods, preferably cancer cells orneurite growth cones, on a substratum, (b) exposing the attachedpseudopods to a putative agent, and (c) determining the effect of theputative agent on the pseudopods, wherein a significant change inpseudopod attachment indicates the putative agent is an effectiverepellent agent. The pseudopods arc preferably attached to thesubstratum by first diluting the pseudopods 1:1 into 2×modified Krebsbuffer containing 22 mM HEPES buffer, pH 7.2 and a reagent to increaseosmolarity, preferably 22 mM sucrose, and then by spinning the solidsupport containing the substratum for a sufficient speed and time tofacilitate attachment, preferably about 2000×g to about 10,000×g, for upto 60 minutes, and more preferably about 5000×g for 15 minutes at roomtemperature.

Various methods for determining the effect of the putative agent on thepseudopods include, for example, monitoring pseudopod detachment,pseudopod elongation, pseudopod retraction, cell extension, or adhesionsites. In one embodiment, the amount of pseudopods detached from thesubstratum is measured. In these methods, a significant amount ofdetached pseudopods indicates the usefulness of the putative agent as arepellent. Particularly useful repellents will detach at least 35%,preferably at least 50%, and most preferably at least 90% of thepseudopods from the substratum.

In another aspect of the invention, the methods are accomplished byexposing whole cells or components of cells, such as the pseudopodsidentified above, to a putative repellent agent. The ability of theputative agent to activate a parameter of the repellent signalingpathway is then determined. The parameters assayed for activation inthese methods include cytosolic phospholipase A₂ (cPLA₂), 12-lipxygenase(12-LOX) or protein kinase C (PKC). Activated cPLA₂ is measured by theamount of arachidonic acid (AA) produced after exposure to the putativeagent, whereas activated 12-LOX is measured by the amount of either12-hydroperoxyeicosatetraenoic acid or preferably12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Protein kinase Cactivation can be measured by the amount of phosphorylated growth coneproteins, such as phosphorylated MARCKS, MacMARCKS, GAP43, or exogenoussnythetic substrate (poly)peptides, such as the phosphorylation sitedomain peptide (PSD).

The invention further relates to methods of identifyingneurite-promoting agents that inhibit the ability of an endogenousrepellent from affecting cell motility. The methods are accomplished byattaching cellular pseudopods to a substratum, exposing the attachedpseudopods to a putative agent in the presence of a known repellent, andthereafter determining the amount of attached or detached pseudopods. Auseful neurite-promoting agent will have been identified if at least75%, preferably at least 85%, and most preferably at least 95% of thepseudopods remain attached to the substratum. Alternatively, the methodscan be accomplished by determining whether the putative agent inhibitsthe activation of a parameter in the repellent signaling pathway, suchas cPLA₂, 12-LOX or protein kinase C.

The invention also relates to therapeutic methods of using the agentsidentified in the above assays. The methods include inhibiting cellmotility, for example the metastasis of cancer cells, by administeringan effective amount of a repellent agent identified in the assays of thepresent invention. Methods of promoting neurite growth or regenerationare also provided in which a neurite-promoting agent is administered toa patient in need of increased neurite formation.

The invention also provides methods of identifying a repellent receptorin which a thrombin-responsive cancer cell that does not respond to aspecific repellent is transfected with a nucleic acid library from acell that responds to the repellent. The transfected cancer cells arethen exposed to the repellent of interest. If a parameter within therepellent signaling pathway is activated in a transfected cell, thenovel repellent receptor is thereafter identified. The identification ofnovel repellent receptors can lead to the production of repellentagonists or antagonists depending on the desired effect.

A further aspect of the invention relates to methods of using therepellent signaling pathway to develop diagnostic tests for themetastatic potential of cancer cells. The methods involve determiningthe presence of repellents or repellent receptors or determining whetherthe cancer cells contain a functional signaling pathway. Test samplesfrom surgically removed tumors, tumor biopsies or cell smears can beused in these methods to determine the metastatic potential of the tumorcells. The deficiency of any of these markers provides evidence thetumor cells may have increased metastatic potential.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relationship between cell adhesion and motility.

FIG. 2 depicts the signaling pathway regulating cell adhesion.

FIG. 3A. Thrombin activation of growth cone cPLA₂. Isolated growth cones(GCPs) were preincubated with thrombin for 10 minutes on ice. AA releasewas measured with ¹⁴C-AA-PI as substrate in a 10-min assay. The 14-aminoacid, thrombin-receptor-activating peptide (TRAP) also stimulatedcPLA₂-PI 4.3-fold.

FIG. 3B. Thrombin does not activate phospholipase C in GCPs, measured as¹⁴C-diacylglycol (DAG) release from ¹⁴C-AA-PI at different calciumlevels.

FIG. 4. Thrombin activation of growth cones stimulates generation from¹⁴C-AA-PC of AA as well as of a compound co-migrating in thin-layerchromatography with 12(S)-HETE. Data are from the same experiment, withseparate processing of samples for AA and HETE analysis.

FIG. 5. Semaphorin IV activation of growth cone cPLA₂. Assay was as forFIG. 7, but growth cones were incubated with recombinant semaphorin IV.Concentrations are nominal, the concentration of active semaphorin isnot known.

FIG. 6. Thrombin-induced GCP detachment. Isolated GCPs were plated onlaminin, incubated for 10 minutes at 37° C., and unbound material waswashed off. After 20 min incubation with thrombin or TRAP (6-amino acidpeptide) at 37° C., GCPs in the supernatant were collected andquantified by protein assay.

FIG. 7A. Thrombin stimulates MARCKS phosphorylation in a12-LOX-dependent manner. FIG. 7B. 12(S)-HETE stimulates MARCKSphosphorylation by PKC in isolated GCPs. Permeabilized GCPs wereincubated with ³²P-ATP and different concentrations of 12(S)-HETE or itsbiologically inactive stereoisomer, 12(R)-HETE, at 30° C. for 30 secondsonly. Polypeptides were resolved by SDS-PAGE. MARCKS phosphorylation wasquantified by phosphorimaging.

FIGS. 8A-8C. Thrombin activation of phospholipases in MAT-Lu cells. Ashows the release, in the presence or absence of 200 nM thrombin, of AA(phospholipase A₂ activity) and of diacylglycerol (DAG; phospholipase Cactivity) from ¹⁴C-AA-labeled phosphatidyl-inositol (PI), -ethanolamine(PE) or -choline (PC) used as substrates. B shows thrombin-stimulated(200 nM) phospholipase A₂ activity as fold increase above control, inthe presence or absence of the reducing agent dithiothreitol (DTT),which inactivates secreted but not cytosolic enzyme, for the threesubstrates. C shows the dose response of phospholipase A₂ activation bythrombin, as AA release from PE.

FIG. 9. Thrombin activation of cPLA₂ in AT-2 cells incubated with either¹⁴C-AA-PI, -PC or -PE as substrate, for 10 min at 37° C. Released AA anddiacylglycerol were isolated and counted.

FIG. 10. Dose response of thrombin inhibition of cell motility. Boydenchamber assays were run for 4 hrs in serum-free medium, with differentconcentrations of rat thrombin in the lower well. Thrombin at 200 nMalmost completely suppresses cell migration across the membrane.

FIGS. 11A-11B. Whisker-box plots of MAT-Lu pseudopod lengths (measuredfrom the center of the nucleus to the end of each pseudopod) after 15min exposure to different concentrations of arachidonic acid (FIG. 11A)or to 10⁻⁴ M AA with or without pretreatment with the 12-lipoxygenaseinhibitor CDC (FIG. 11B). In these plots the center points of each barindicate the median, the ends of the boxes the 25% and 75% quantiles,and the ends of the whiskers the 5% and 95% quantiles. AA causesstatistically significant pseudopod shortening, which is reversed tocontrol levels by CDC. Plots were generated from the type of raw datashown in FIG. 14.

FIGS. 12A-12D. Whisker-box plots of pseudopod lengths of control cellsand cells exposed for 15 min to different concentrations of various HETEisomers (A,B,C). FIG. 12D shows the results of pretreatment with theprotein kinase C inhibitor, calphostin C, on the effect of 12(S)-HETE.For explanation of the plot, see FIG. 11.

FIG. 13. Pscudopod lengths of AT-2 cells incubated for 15 min at 37° C.in different concentrations of 12(S)-HETE. The Whisker-Box plots showquantiles of pseudopod length; vertical lines, 5 and 95% quantiles;boxes, 25 and 75% quantiles; connecting line, 50% quantile. Note thedifferences between this dose-response curve and that for MAT-Lu cells,shown in FIG. 12A.

FIGS. 14A-14B. Histograms of pseudopod lengths of MAT-Lu cells exposedto different concentrations (M) of 12(S)-HETE for 15 min. FIG. 14A showsthe control and concentrations of 12(S)-HETE ranging from 10⁻¹²M to10⁻¹⁰M. FIG. 14B shows concentrations of 12(S)-HETE ranging from 10⁻⁹Mto 10⁻⁶M. The distribution patterns were fitted by gamma regression, andthe vertical line indicates the mode. The same data are also shown as awhisker-box plot in FIG. 12A.

FIG. 15. Time course of pseudopod shortening induced by 10⁻¹⁰ M12(S)-HETE introduced at time 0 by medium replacement. Data were pooledfrom 73 identified pseudopods (on 37 cells) followed over 20 min.Pseudopod lengths reach a minimum at about 17 min. For explanation ofwhisker-box plot, see FIG. 13.

FIG. 16. 12(S)-HETE effect (15-min incubation) on the pseudopod lengthsof low-motility (AT-2) and high motility (MAT-Lu, MAT-LyLu and AT-3)Dunning carcinoma cells. 50% quantiles are plotted. Note the differentresponses of the two classes of cells

FIG. 17. Quantitative analyses of growth cone collapse induced bythrombin, TRAP or 12(S)-HETE, with or without CDC pretreatment. Collapsestatus was assessed on fixed neural cultures having undergone thefollowing treatments: Control, vehicle alone; thr, thrombin (100 nM) for7 minutes; thr/CDC, CDC (10 μM) pretreatment for 30 minutes, followed bythrombin (100 nM) for 7 minutes; TRAP (100 mM) for 7 minutes; TRAP/CDC,30 minutes CDC (10 μM) pretreatment followed by TRAP (100 mM) for 7minutes; 12(S)-HETE (10⁻⁷ M) for 10 minutes. For each condition at least50 growth cones were scored as described in Methods. Data were obtainedfrom at least 2 independent experiments and are presented as percent oftotal growth cones observed.

FIG. 18. Thrombin-induced detachment of GCPs from a laminin substratum.GCPs plated on laminin were exposed for 20 minutes to differentconcentrations of thrombin, with or without cytochalasin D (CD)pretreatment, or to TRAP. Detachment was measured as percent ofpelletable protein collected in the supernatant. In control conditionsdetachment was 17 percent of total GCPs plated. Data shown are increasesin detachment relative to control incubation.

FIGS. 19A-19B. Dose Response curves of PLA₂ activation by thrombin (A)and TRAP (B) in GCPs. GCPs were incubated for 10 minutes on ice withvarying concentrations of thrombin (A) or TRAP (B), then for 10 minutesat 37° C. in the reaction mixture, in the presence of 10 μM CaCl₂. Thesubstrate was ¹⁴C-AA-PI. The data presented are in triplicate. The errorbars represent s.d.; where not present error bars were too small to beindicated.

FIG. 20. Effects of calcium on control and thrombin-stimulated levels ofPLA₂ in GCPs. GCPs were incubated alone (control) or in the presence of100 nM thrombin for 10 minutes on ice, then combined with substrate(¹⁴C-AA-PI) for 10 minutes at 37° C., in the presence of EGTA or varyingμM concentrations of CaCl₂ (“0 Ca” indicates no addition of Ca²⁺ orEGTA). AA release was measured as described in Methods. Data representseveral experiments, each done in triplicate. Error bars are s.d.

FIG. 21. Substrate selectivity of thrombin-stimulated growth cone PLA₂.GCPs were assayed in the absence (control) of presence of 100 nMthrombin (thr) with 10 μM CaCl₂ and equal concentrations of either¹⁴C-AA-PI, -PE, or -PC as substrate. Numbers above thrombin columnsindicate increase in activity relative to control levels. Data areaverages of 3 experiments, all done in triplicate, error bars indicates.e.m.

FIG. 22. Thrombin inhibits growth cone PLC in a calcium-dependentmanner. GCPs were incubated alone (control) or in the presence of 100 nMthrombin for 10 minutes on ice, then combined with substrate (¹⁴C-AA-PI)for 10 minutes at 37° C., in the presence of EGTA or varying μMconcentrations of CaCl₂ (“0 Ca” indicates no addition of Ca²⁺ or EGTA).DG release was measured as described in Methods. Data represent severalexperiments, each done in triplicate. Error bars are s.d.

FIG. 23. IGF-1 stimulation of PLC and PLA₂ in growth cones. GCPs wereassayed for both PLA₂ and PLC activity as described, in the presence of10 μM CaCl₂ and varying concentrations of IGF-1, using ¹⁴C-AA-PI assubstrate and recovering ¹⁴C-AA and ¹⁴C-AA-DG as products. Data areaverages of several experiments, each done in triplicate. Error barsindicate s.d. Where no error bars appear s.d. was too small to register.

FIGS. 24A-24C. Thrombin-stimulated release of AA and HETE. GCPs wereassayed for both PLA₂ and Lox activity as described. Inhibitor (0.156 μMor 0.2 μM indomethacin) was introduced to some GCP samples for 5 minuteson ice prior to the addition of 100 nM thrombin. Following an additional10 minutes incubation on ice, the GCP mixture was then incubated witheither ¹⁴C-AA-PC (A,B) or ³H-AA (C), in the presence of 10 μM CaCl₂ for5 minutes at 37° C. A and B show thrombin-stimulated release of AA andHETE in assays using the PC substrate, as actual amounts (A) and foldincrease (B). C illustrates thrombin stimulation of HETE directly fromthe AA substrate. In A and B, values for control and thrombin-stimulatedconditions were averaged from 6 experiments, all done in triplicate,while the values for CDC and indomethacin-treated GCPs were averagedfrom 2 experiments, done in triplicate. Data in C are from onerepresentative experiment, done in triplicate. Error bars indicate s.d.;where not seen, error bars were too small to be indicated.

LC/MS² was used to demonstrate GCP synthesis of specific HETE isomer(s).FIGS. 25A-D show HPLC elution patterns. The abscissa indicates relativedetection levels of fragments derived from HETE isomers (negative ionm/z 319.3) and characteristic of 12-HETE (fragment m/z 179.1) (FIGS. 25Cand 25D) and 15-HETE (fragment m/z 219.1) (FIGS. 25A and 25B),respectively. FIGS. 25B and 25D show corresponding deuterated standards,which yield fragments of greater mass. Based on scale and peak width,12-HETE is the predominant species, but a significant level of 15-HETEis evident. 5-HETE was not detected.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the elucidation of areceptor-activated signaling pathway that leads to cell detachment andeventual inhibition of cell motility. As shown in FIG. 2. the pathwaystarts with the stimulation of cytosolic phospholipase A₂ (cPLA₂) when arepellent molecule activates a repellent receptor. The stimulation ofcPLA₂ produces high levels of endogenous arachidonic acid (AA). AA isthen converted by 12-lipoxygenase (12-LOX) into 12(S)- and some15(S)-hydroxyeicosatetraenoic acid (12(S)-HETE and 15(S)-HETE,respectively) which, in turn, activates protein kinase C at adhesionsites. The substrates for protein kinase C include MARCKS, MacMARCKS andGAP43. The phosphorylation of MARCKS, MacMARCKS or GAP43 leads toloosening of the adhesion site, resulting in either pseudopod spreadingor, in the extreme case, pseudopod detachment. Concomitantly, theaffinity of adhesion molecules for their extracellular ligand also maybe reduced. Therefore, pseudopod repellents decrease cell adhesion thatcan lead to either increased motility with weak stimulation or celldetachment and repulsion with strong stimulation.

Non-metastatic cells respond to repellents via the signaling pathwaywhich prevent them from moving out of a tissue. For example, neuritegrowth may be inhibited by these repellents. In contrast, metastaticcancer cells ignore the repellents because they lack the functionalreceptor and/or have defects in the signaling pathway. The presentinvention relates to the identification of agents that can regulate thispathway.

A. Repellent Assays

One aspect of the invention relates to methods for identifying an agentthat promotes significant cell detachment and inhibits the motility of acell. The assays are generally accomplished by:

(a) attaching cellular pseudopods to a desired substratum;

(b) exposing the attached cellular pseudopods to a putative repellentagent; and

(c) determining the effect of the putative agent on the pseudopods,wherein a significant change in pseudopod attachment indicates theputative agent is an effective repellent agent.

As used herein, the term “cellular pseudopods” includes any cell or cellcomponent that can attach to a substratum and contains a functionalrepellent signaling pathway, including, for example, neuronal cells,cancer cells and hybrids of repellent-responsive cancer cells describedbelow. In addition, cellular pseudopods can also be constructed throughrecombinant or transfection technology in which genes encoding acomponent involved in the pathway, for example, a desired repellentreceptor, can be inserted into host cells according to methods known inthe art.

Growth cones (tips of growing neurites) are particularly useful in theseassays. They can be isolated from fetal or newborn brains as described,for example, in Pfenninger ct al., Cell 35:573-584 (1983), incorporatedherein by reference. The growth cones can also be derived from othercellular sources, for example, cultures of growing neuronal cells.

Cancer cells, especially highly motile cancer cells, are also useful inthese assays. The cells can be obtained from a primary source such assurgically removed tumors, tumor biopsies or cell smears. Alternatively,cultured cancer cells can be grown and harvested for this purpose. Inthis regard, highly motile prostate carcinoma or melanoma cells areparticularly useful in these assays.

Hybrid cells can also be produced in these assays. These cells can beconstructed from a first cell (cell type X) that has a receptor for aputative repellent and a second cell (cell type A) that responds fullyto a known repellent (for example, thrombin), indicating that thesignaling pathway is functional in cell type A. The XA hybrid cells canthen produced according to methods known in the art and contain theputative repellent receptor contributed by cell type X and thefunctional signaling pathway contributed by cell type A. The hybrids canthen be used in the assays of the present invention to determine whetherthe putative repellent activates the signaling pathway as shown by celldetachment.

In the first step of the assay, the cellular pseudopods are attached toany desired substratum according to any method known in the art or asdescribed in the examples below. Preferably, the cellular pseudopods arcplated on substrata that are composed of extracellular matrix moleculesor cellular adhesion molecules that are attached to a solid support,such as plastic culture dishes and the like. The solid support isoptionally pre-coated with nitrocellulose or other similar coating priorto the addition of extracellular matrix molecules or cellular adhesionmolecules. Extracellular matrix molecules useful in these methodsinclude, for example, laminin, fibronectin, collagen or any combinationof these proteins. Cellular adhesion molecules can include, for example,L1, N-CAM, cadherin, glycolipids, oligosaccharides, or syntheticpolypeptides of these molecules, as well as any combination of thesecellular adhesion molecules.

The culture dishes or other solid support can be centrifuged for asufficient time and at a sufficient speed to facilitate adhesion siteformation. The solid support can be centrifuged at a speed from about2,000×g to about 10,000×g for up to 60 minutes, preferably at about5,000×g for about 15 minutes at room temperature.

The pseudopods arc then rinsed in an appropriate buffer in order toseparate the adherent pseudopods from the non-adherent pseudopods.

In the next step of the assays, the attached pseudopods are then exposedto potential repellent molecules or preparations. Such preparations maybe extracts of adult or developing tissues and subfractions thereof, orpurified or synthetic (poly)peptides or other factors, for example,cytokines. growth factors or neurotransmitters.

In these and other methods described below, effective repellents can beidentified qualitatively by examining their effect on therepellent-signaling pathway, for example, by monitoring pseudopoddetachment, pseudopod elongation and/or retraction, cell extension,adhesion sites, or other significant change in pseudopod attachment.Methods that can be used to monitor such activity are provided in theexamples below and include, for example, the Boyden chamber assay, thepseudopod length assay, various microscopic techniques and, asidentified above, various protein assays.

In one embodiment, methods for identifying repellents by monitoringpseudopod detachment are generally accomplished by:

(a) attaching cellular pseudopods to a desired substratum;

(b) exposing the attached cellular pseudopods to a putative repellentagent; and

(c) determining the amount of cellular pseudopods detached from thesubstratum.

In these methods, the detached pseudopods are recovered from thesupernatant by any method known in the art or as described in theexamples below. Preferably, the detached pseudopods are centrifuged toisolate them from the supernatant. The detached pseudopods can then bequantified by any protein assay known to those skilled in the art,including, for example, immunological assays. In these methods, aputative agent is an effective repellent if at least 35%, morepreferably at least 50%, and most preferably at least 90% of the testcells are detached from the substratum, resulting in reduced cellmotility.

Effective repellents can also be identified by monitoring the up-ordown-regulation of genes. Methods for measuring gene expression includedirect measurement of products of transcription and translation, i.e.,mRNA or protein, by means well known in the art, including for example,by the use of microarrays, PCR, Rnase protection, enzyme andimmunoassays. The effects of various compounds on the individual genesencoding components of the pathway also can be monitored by attaching aregulatory region of such a gene to a reporter gene, for example, CAT orluciferase, and monitoring expression of the gene upon exposure of thegene construct to a compound of interest.

In addition, genes encoding protein repellent agents can be used todetermine alterations in endogenous gene homologs. These alterationscould result in decreased and/or altered gene activity and efficacy.Smaller sequences of the gene can also be used as probes in suchdiagnostic methods. Genes and proteins encoded thereby, and modifiedversions thereof, can be used to generate antibodies or other probesthat are useful as diagnostic agents and potential therapeutic agents.

In a further embodiment of the invention, methods for identifyingrepellent agents are provided in which parameters within the signalingpathway are monitored. The methods are generally accomplished by:

(a) obtaining a whole cell or component thereof having a functionalrepellent signaling pathway;

(b) exposing the whole cell or component thereof to a putative repellentagent; and

(c) monitoring a parameter on the repellent signaling pathway, whereinthe putative repellent agent inhibits cell motility if the parameter isactivated.

As an example of these methods, growth cones (isolated and functionallyintact), metastatic cancer cells or the hybrid cells described above areexposed, either in suspension or attached to a substratum, to differentrepellent factors or their secondary messengers, and a parameter in thesignaling pathway is monitored for activation. A parameter is monitoredaccording to methods known to those skilled in the art or as describedin the examples below and include, for example, measuring the number andaffinity of repellent binding sites, using radiolabeled (preferably ¹⁴Cor ³H) repellent or receptor antibodies, and/or by monitoring the enzymeactivities involved in the signaling cascade. These parameters include:(1) different forms of cytosolic phospholipase A₂ [calcium-dependentor—independent; measured as arachidonic acid release from differentphospholipid substrates, for example, phosphatidylcholine (PC),phosphatidylethanolamine (PE) or phosphatidylinositol (PI)]; (2)different forms of 12-lipoxygenase, measured as production of 12(S)-HETEfrom arachidonic acid or different phospholipids; and (3) various formsof protein kinase C, measured by phosphorylation of growth coneproteins, such as MARCKS, MacMARCKS, GAP43, or exogenous substrates,such as PSD peptide.

The agents identified by the assays can be used as anti-invasive andanti-metastatic agents for the treatment of cancer cells that aremetastatic or susceptible to metastasis. The anti-metastatic agent canbe formulated in any pharmaceutically acceptable formulation in anypharmaceutically acceptable form. Such forms and formulations includeliquids, powders, creams, emulsions, pills, troches, suppositories,suspensions, solutions, and the like. Thrombin and semaphorin are usedin preferred embodiments in accordance with the method of the instantinvention. Therapeutically effective amounts of anti-metastaticmolecules can be any amounts or doses that are sufficient to bring aboutthe anti-metastatic effect and depend, for example, on patient size andcondition and type and location of the cancer. The dosages can be givenas a single dose, or as divided doses, for example, divided over thecourse of several weeks. The anti-metastatic molecules of the instantinvention can be administered by any suitable means, including orally orby injection. In the preferred embodiment of the invention, the agent isadministered by injection. Such injection can be locally administered toany affected area. For example, thrombin, thrombin receptor-activatingpeptide (“TRAP”), semaphorin or other repellent can be injectedurethroscopically into the prostate.

Modified forms of the agent having increased activity can also bedelivered therapeutically. Such modified forms include, but are notlimited to, more limited functional domains, site-specific modificationsthat increase efficacy or activity in functional assays, and modifiedagents in which functional domains, for example, from humans or otherspecies are combined with effector domains such that species-specificsignals can be used to elicit protein activity.

B. Assays for Neurite-Promoting Agents

As previously noted, the invention also relates to the identification ofneurite-promoting agents. Evidence indicates that nerve regeneration inthe CNS, for example, after spinal cord injury, is inhibited byendogenous repellents. Therefore, agents that block a repellent frombinding to its receptor or inhibit a parameter further downstream in thesignaling pathway can promote nerve regeneration after injury.

The methods of this aspect of the invention are generally accomplishedby:

(a) attaching cellular pseudopods having a functional signaling pathwayto a substratum.

(b) exposing the attached cellular pseudopods to a putativeneurite-promoting agent; and

(c) determining the effect of the putative agent on the cellularpseudopods, wherein a putative agent is a neurite-promoting agent if arepellent is blocked from binding its receptor or the signaling pathwayis inactivated.

The method is generally performed as described in the above methods foridentifying repellents. Neurite growth cones are particularly useful inthese assays and can be derived from neural tumor cells or primaryneurons. Neurite-promoting agetns to be tested may be extracts fromadult or developing tissues and subfractions thereof, or purified orsynthetic (poly)peptides or other factors, for example, cytokincs,neurotransmitters or growth factors. They also may be reagentsinterfering with the signalling pathway, such as LOX, PLA₂ or PKCinhibitors

The effectiveness of the putative agent can be determined by a number ofmethods. In one method, a qualitative or quantitative measure ofeffectiveness can be determined by the amount of detached pseudopods, orconversely the amount of pseudopods that remain attached to thesubstrate. In this regard, an effective neurite-promoting agent willhave been identified if at least 75%, preferably at least about 85% andmost preferably at least about 95% of the pseudopods remain attached tothe substratum.

Alternatively, the inhibition of the repellent-stimulated signalingpathway can be determined by assaying for cPLA₂, 12-lipoxygenase orprotein kinase C activity as described above. If desired, effectiveinhibitors can then be further tested in neurons or neural tumor cells,such as PC 12, growing in culture to assess whether they can inhibit theeffect of specific repellents. This assessment can be done by measuringneurite length and/or direction of neurite growth relative to therepellent source.

The neurite-promoting agents of the present invention can be used totreat disorders caused by the lack of nerve growth or regeneration, forexample, spinal cord injury. Those skilled in the art can readilydetermine a therapeutically effective dose and mode of administrationdepending on various factors, including, for example, the location andextent of need for neurite generation and patient size. Preferably, theneurite-promoting agent is administered by injection.

C. Identification of a Repellent Receptor

The present invention additionally provides methods for identifyingnovel repellent receptors that include the following steps:

(a) selecting a known repellent-responsive cancer cell that does notrespond to a second repellent;

(b) transfecting the cancer cell with a nucleic acid from a cell thatresponds to the second repellent;

(c) cloning and culturing the transfected cancer cell with the secondrepellent;

(d) measuring the amount of an activated parameter in the transfectedcancer cell; and

(c) identifying the repellent receptor encoded by the nucleic acid.

For example, thrombin-responsive cancer cells that do not respond to aspecific repellent, such as semaphorin IV, are transfected with a cDNAlibrary from a cell type responding to this repellent (e.g., semaphorin)and cloned. Cloned transfectants are exposed to the specific repellentand assayed for cPLA₂ activation. The cDNA insert in responding cells isused to identify the novel repellent receptor. This is an expressioncloning approach. Cancer cells that contain the signaling cascadedescribed above and properly express the transfected receptor to therepellent are expected to increase cPLA₂ activity when incubated withthe repellent. Therefore, the cPLA₂ assay can be used to screen tiecells transfected with the appropriate cDNA library. Once cloned, thetransfected insert in responding cells can be isolated by any meansknown in the art, including, for example, by fluorescence activated cellsorter (FACS), and analyzed to identify the receptor.

The identification of such receptors is desirable because they can beactivated to block cancer cell metastasis or inhibited in the CNS topromote nerve regeneration. The receptors can be used in screeningmethods well known in the art to find compounds that bind thereto toproduce the desired effects. Cells expressing the receptor to arepellent or immobilized isolated receptor can be screened for bindingof peptides or other chemicals that may interfere with repellent binding(i.e., antagonists). In assays involving cells or cell membranes, suchreagents can be tested for activation or inhibition (in the presence orabsence of repellent) of the repellent receptor, using the cPLA₂ assayor other downstream parameters.

D. Metastatic Potential of Cancer Cells

The invention also provides methods of using the repellent signalingpathway to determine the metastatic potential of cancer cells. Themethod is based on a deficiency of repellent molecules or receptorsand/or when one or more of the signaling steps (i.e., parameters) in thepathway is deficient.

Accordingly, the methods involve:

(a) obtaining, a specimen of the cancer cells; and

(b) determining a deficiency of;

(i) repellent in the specimen;

(ii) repellent receptors in the cancer cells; or

(Iii) a functional repellent signaling pathway in the cancer cells.

A deficiency in any of (i)-(iii) indicates the cancer cells haveincreased metastatic potential. Such deficiencies can be determined by:(1) the expression of different repellents, for example semaphorins orthrombin, in the neighborhood of the tumor; (2) the expression of therepellent receptor in the tumor; (3) the expression of gene products ofthe signaling pathway, such as specific forms of cPLA₂ specific forms of12-lipoxygenase, specific forms of protein kinase C; (4) the enzymaticactivity of these gene products; and (5) the expression of MARCKS,MacMARCKS, or GAP43. Such deficiencies are markers of metastaticpotential.

Suitable assays for the methods include, for example, Northern blot ofgel transfers or spotted extracts, polymerase chain reaction (PCR) ofDNA or RNA (with prior reverse transcription), Rnase protection assayand/or in-situ hybridization to determine the presence of particularmutations in, or the expression levels of, specific genes involved inthe signaling pathway, its receptor or the repellent that activates it.Additional assays include immunological methods to determine the levelsand distributions of the gene products in the cancer cells. Such testscould be, for example, immunoblots of gel transfers or of spottedprotein samples, immunoelectrophoresis, or immunocytochemistry ofsectioned tissues or cell smears. Finally, enzyme activities (such as12-LOX or protein kinase C) can be measured in cell homogenates with orwithout stimulation with putative repellents. Such assays would make useof exogenous, radiolabelled substrates. The test specimens can besurgically removed tumors, tumor biopsies or cell smears. Tissues andcells can be processed for histologic/cytologic examination and/orhomogenization, DNA, RNA or protein extraction. The homogenates orextracts can be analyzed by the biochemical methods listed above.

A panel of tests measuring mis-expression of the various proteinsinvolved in the signaling pathway (e.g., repellent, repellent receptor,cPLA₂, 12-LOX, PKC isoforms, MARCKS, MacMARCKS, GAP43) is particularlyuseful. The application of such a panel, via a combination of assays,would provide the strongest evidence of whether a particular cancer haslost its responsiveness to repellents and, therefore, may have increasedmetastatic potential. Such a panel can include, for example, activationof cPLA₂ with known repellents, PCR to test for the presence of specificmutations in the 12-LOX gene, immunochemistry to determine whether aparticular protein kinase C isoform is absent from tumor cells (whilepresent in normal cells).

The following Examples are intended to illustrate, but not limit, thepresent invention.

EXAMPLE 1 Experimental Systems

In several of the examples belong Dunning rat prostate carcinoma cellsthat have been subcloned into two types of cell lines were used in thestudies. The first type include cell lines of high metastatic potentialand high motility (MAT-Lu, MAT-LyLu and AT-3), whereas the second typeinclude cell lines of low metastatic potential and low motility (AT-1and AT-2). Both are described in Isaacs et al., Prostate 9:261-281(1986). The cell lines were used to study whether metastatic cancercells share some of the molecular characteristics of nerve growth cones.The phenotypes of both types of cell lines are strikingly different.AT-1 and AT-2 cells exhibit typical clonal (i.e., clustered) growth inculture, Whereas the MAT-Lu, MAT-LyLu and AT-3 cells are highly motileand spread rapidly all over the tissue culture dishes. The two cellclasses were compared to growing neurons (i.e., growth cones), takingadvantage of the rat origin of both systems.

In addition, seven human prostate carcinoma cell lines were used (seeTable 2). One or two cell lines of medium to high motility (e.g.,PC-3M-MM2 and DU145) are first selected. The PC-3M-MM2 cells disperserapidly in the dish and, like the MAT-Lu cells, never form colonies.Cells are grown according to standard procedures. Large numbers ofaliquots of all cancer cell lines arc collected and frozen in order toavoid the use of higher passage numbers in later experiments. As theexperimentation progresses, the motility and morphology of the cells isre-checked to avoid distortion of results by changes in phenotype. Forexperimental use, cells are kept well below confluence. In order to testfor potential effects of the growth matrix, cells are grown for selectedexperiments not only on serum-conditioned tissue culture plastic butalso on laminin, collagen, fibronectin or Matrigel (LifeTechnologies/GIBCO-BRL, Gaitheresberg, Md.; BectonDickinson/Collaborative Biomedical Products, Bedford Mass.). Experimentswith polypeptides factors are performed with cells that have been grownfor a few hours in low-serum (1%) or serum-free medium supplemented withpure bovine serum albumin. The motility, of all cells is assessedquantitatively using the (long-term) Boyden chamber assay as describedin Boyden, J. Exp. Med. 115:453-466 (1962), incorporated herein byreference. The assays are performed with serum preconditioned, laminin-,or fibronectin-coated membranes, whose pores (8-10 μm) remain open (vs.those coated with Matrigel). The relative number of cells moving within3-6 hours across the membrane from the upper well is counted in thepresence or absence of IGF-1 in the lower chamber.

To study short-term chemotactic responses of the prostatic carcinoma(CaP) cells to the different agents, the Zigmond chamber, designedoriginally to study leukocyte chemotaxis and available commercially fromNeuroProbe, Inc.(Cabin John, Maryland) is used. These chambers enablethe exposure of cells grown on a coverslip (coated with differentmatrices) to a defined chemical gradient and to study within 15-30minutes cell orientation in this gradient as described in Zigmond. J.Cell Biol. 75:606-616 (1977), incorporated herein by reference. Cellmorphology is recorded digitally at different, defined points of thegradient (dependent on concentration range, distance from the higherconcentration well, and time after establishment of the gradient;gradients last for about 90 min). The cells' longest axis andorientation is determined, and the angles of these axes relative to thegradient's axis are measured and plotted.

EXAMPLE 2 Repellent Effects on Adhesion Sites and Cell Motility

In this example, the effects of repellent or downstream effectors (AA or12(S)-HETE) were examined on pseudopod motility, in the presence orabsence of the appropriate inhibitors. The Zigmond and Boyden assays areused to look at both short- and long-term responses of the cells to thevarious agents. The pseudopod length assay also is used to determineshort-term effects of the various reagents on the cells. Interferencereflection microscopy (IRM) is used to image directly and measure thechanges in adhesion sites formed by live cells, under differentexperimental conditions. Therefore, we can examine quantitativelywhether and at which concentrations the repellents—or down-streameffectors of the cascade—change adhesion sites of CaP cells.

Psezudopod behavior. Pseudopod activity is studied in two differentassays: (i) Long-term (2-5 hrs) effects of repellent on motility,assessed in the Boyden chamber (repellent in the lower well, with orwithout IGF-1 added to the lower chamber). As stated earlier, the Boydenchamber membranes are pre-treated either with laminin, collagen,fibronectin or the like and then with serum. (ii) For short-termanalysis, CaP cell orientation is studied and quantified in the Zigmondchamber. For the Zigmond assay, CaP cells are grown at low density onlaminin or fibronectin-coated coverslips and then placed upside-down ongradients of different factors. After 15 to 30 min, orientation in thegradient is determined (Zigmond, 1977). By adding different but uniformlevels of repellent to a gradient of IGF-1, its influence on pseudopodorientation can be measured and expressed as a dose response. Inaddition, repellents themselves may orient the cells in a gradient ofthe appropriate concentration range, and the system can be used todetermine this possibility. These assays are carried out with differentconcentrations of repellent and, as controls, boiled repellent. Forthrombin, a positive control is TRAP 6, the peptide SFLLRN (Feng et al.,1995). The Zigmond assay also is performed with different concentrationsor gradients of the intermediates, AA and 12(S)-HETE. Other fatty acidsand HETEs (1 2(R)-, 5(S)- and 15(S)-) serve as controls. In addition,the effects of cPLA2, 12-LOX and PKC inhibitors on cell motility ororientation are assessed in the presence or absence of repellentstimulation (cf. FIG. 2). To assess pseudopod behavior further, theZigmond assay can be complemented with the pseudopod length assaydescribed earlier.

Adhesion site microscopy. Adhesion sites can be imaged in live cells byinterference reflection microscopy (IRM) as described in Izzard andLochner, J Cell Sci 21:128-159 (1976); Izzard and Lochner, J Cell Biol42:81-116 (1980); Bereiter-Hahn et al., J Cell Biol 82:767-779 (1979).Changes in adhesion sites in response to repellents and to intermediatesof the cascade are analyzed. Cells arc grown on coverslips coated withlaminin, collagen, fibronectin or the like and placed upside-down on aZigmond or custom-made perfusion chamber. These are examined with anupright Zeiss microscope with IRM optics. In different experimentalconditions, high-resolution images are captured with a Nikon/Kodak1000×1000 CCD camera (linked to an Apple PowerMac 9500/132 MHz computer)and stored for later processing. Because the focal adhesions stand outwith high contrast, dark against a very light background, the images areprocessed by contrast enhancement and background subtraction to obtainon/off images of adhesions. The computer measures the focal adhesionareas and their changes as a function of time. Thus, live cells can beanalyzed in real time to see the changes induced in focal adhesions byrepellents or 12(S)-HETE. These data are useful in conjunction with thebiochemical results obtained in Example 6.

This example provides a tight correlation between repellent activationof the different steps of the cascade and changes in pseudopod behavior,especially, pseudopod detachment. The use of up to four different assaysof pseudopod activity, together with dose-responses of the differentintermediates of the cascade, provides insights into the precise mode ofaction of the repellent.

EXAMPLE 3 Lipid Messengers in Nerve Growth Cones

Viable nerve growth cones can be isolated in bulk from fetal rat brain(Pfenninger et al., 1983). Such isolated nerve growth cones exhibit ahigh metabolism of phosphatidylinositol (PI) and phosphoinositides asreported in Pfenninger et al., The Nerve Growth Cone pp. 111-123 (RavenPress, N.Y. 1991). The primary pathway of PI metabolism is via PLA₂(Negre-Aminou and Pfenninger, J. Neurochem. 60:1126-1136 (1993); andNegre-Aminou et al., J. Neurochem. 67:2599-2608 (1996)). Growth conescontain two high molecular weight forms of cPLA₂ that are selective forPI and PE, respectively, and calcium-independent. However, the enzymesappear to be recruited to the plasma membrane in the presence ofcalcium. At least the PI-selective enzyme (cPLA₂-PI) is likely to benovel (Negre-Aminou et al., 1996). cPLA₂-85 (PE- and PC-selective) isexpressed only at very low levels in growth cones as determined byenzyme assay and Western blot (Negre-Aminou et al., 1996).

Thrombin (100 nM), a potent growth cone repellent as discussed above,stimulates cPLA₂-PI and cPLA₂-PE as well as a PC-hydrolyzing cPLA₂ about5-, 7- and 7-fold respectively (FIG. 3), as measured by release of¹⁴C-AA. Thrombin's effect on growth cone attachment was tested in acell-free assay involving isolated, viable growth cones plated onlaminin. Upon exposure to the same concentrations of thrombin used forthe cPLA₂ assays, about 46% of growth cones detached, compared to 17%for controls, but only in the presence of calcium (FIG. 6). The cPLA₂activation and growth cone detachment responses could be mimicked(although much less effectively) with proteolytically inactive,thrombin-receptor-activating peptides (TRAPs) described in Feng et al.,J. Med. Chem. 38:4125-4130 (1995) and Grand et al., Biochem. J.313:353-368 (1996). These results indicate the involvement of a thrombinreceptor. Other data indicate dose-dependent activation of growth conecPLA₂ by another repellent, recombinant SemaIV (FIG. 5). The repellentnetrin (recombinant) also stimulates the enzyme, at least weakly.

While much of the AA liberated by cPLA₂ is reincorporated intophospholipids in growth cones (Negre-Aminou ct al., 1993), some of it ismetabolized by endogenous 12-LOX to 12(S)-HETE. During incubation ofgrowth cones with ¹⁴C-AA (60 min, 37° C.), approximately 5% of the labelis converted into 12(S)-HETE, and this is blocked by the LOX inhibitornordihydroguaiaretic acid (NDGA). More significantly, growth conesincrease generation of a compound co-migrating in TLC with 12(S)-HETEand sharing its molecular mass from ¹⁴C-AA-labelled phospholipid 4-8fold relative to controls when stimulated with thrombin. Consistent withthese results, Western blots of growth cone polypeptides probed forleukocyte-type 12-LOX with antibody exhibited a single immunoreactiveband. These data indicate that growth cones contain a 12-LOX related oridentical to leukocyte 12-LOX, and that stimulation with a repellentsuch as thrombin actually increases 12(S)-HETE levels in the growthcone.

The major putative target of 12(S)-HETE in growth cones is proteinkinasc C (PKC), which is highly enriched in nerve growth cones. Isoformsof 12(S)-HETE present in growth cone adhesion sites are PKC βI, γ, ε, ι,λ and traces of θ. PKC phosphorylates two major substrates in nervegrowth cones, the neuronal protein, GAP43, and MARCKS or the relatedMacMARCKS (Katz ct al., J. Neurosci. 5:1402-1411 (1985); Meiri ct al.,Proc. Natl Acad sci USA 83:3537-3541 (1986); Skene, Ann Rev Neurosci12:127-156 (1989); and Stumpo et al., PNAS,USA 86:4012-4016 (1989)). Itwas observed that thrombin stimulates MARCKS and GAP43 phosphorylationin isolated growth cones and that this is dependent upon PKC as well as12-LOX activity (inhibition of these enzymes with calphostin or CDC,respectively, blocks phosphorylation (FIG. 7A). 12(S)-HETE stimulationof PKC in nerve growth cones was also examined. In short timephosphorylation assays (30 sec @ 30° C.), at 10⁻⁶M or less free Ca²⁺,12(S)-HETE (but not its stereoisomer, 12(R)-HETE) stimulates MARCKS andGAP43 phosphorylation in a dose-dependent, biphasic manner peaking atabout 10⁻¹⁰M (FIG. 7B). Naor et al. Mol. Endocrinology 2:1043-1048(1988) observed a similar, biphasic response of PKC γ to AA, with thepeak at 12 μM. The rapid and low-dose response we observe is notcompatible with the involvement of a 12(S)-HETE receptor (with a K_(d)of 10⁻⁹M and requiring 90-120 min for saturation) and of a complex,intervening signaling pathway as suggested by Liu et al., PNAS, USA92:9323-9327 (1995).

The growth cone studies herein indicate that: (a) the repellent thrombincauses cPLA₂ activation and growth cone detachment, and (b) that therepellent SemaIV seems to operate through the same pathway as suggestedby its strong activation of cPLA₂. (c) Thrombin also raises growth cone12(S)-HETE levels and (d) phosphorylation of MARCKS (and/or MacMARCKS)and GAP43. Furthermore, (e) 12-LOX and PKC activity are necessary and(f) 12(S)-HETE sufficient for the repellent effect.

Dunning MAT-Lu cells exhibit a highly motile phenotype as mentionedabove. In Boyden chamber assays (migration across a laminin-coatedmembrane with 8-μm pores over a 4 hr period), MAT-Lu cells readily crossthe barrier, and this is enhanced about 1.8-fold by IGF-1 in the lowerchamber. Control motility and positive chemotaxis to IGF-1 are inhibitedby thrombin added to the lower chamber (Table 1 and FIG. 10). Whenthrombin is added to the cells in a regular culture dish, pseudopods areseen to retract within 5-10 min. Thus, thrombin (at the concentrationsused) acts on these cells' pseudopods as a negative-clemotactic orrepellent factor. The cells are plated in culture dishes in the presenceof serum. Several hours later medium is replaced by serum-free medium.Putative repellent factors, such as thrombin, are added and the culturesincubated for 5-10 min. The cultures are then examined in the invertedmicroscope and compared to control cultures. Cells are photographed andthe photographs digitized, or they are digitized directly, and thestored images are analyzed by measuring pseudopod lengths between theirtips and the nucleus (see FIG. 14). This pseudopod retraction assay canbe used to screen for repellents and negative-chemotactic actors thatmay be anti-metastatic factors.

TABLE 1 Effects of IGF-1 and Thrombin on Cell Migration* Control IGF-1(0.75 nM) Control 99.8 +/− 5.9 184 +/− 15 Thrombin 30.9 +/− 5.7  38 +/−9.2 (200 nM) CDC 97.5 +/− 11.0 118 +/− 10.4 (126 nM) Thrombin +  107 +/−22.0 Not done CDC *Mat-Lu cells crossing filter in % of control +/− SEM

When cytosolic or membrane fractions of MAT-Lu cells are assayed forcPLA₂ activity (using ¹⁴C-AA-labeled PC, PE or PI as substrates), AArelease from PE is readily measurable whereas PC and PI are poorsubstrates. In the prostate, most AA appears to be in PE (Pulido et al.1986). In 10-min assays, exposure of MAT-Lu cells to thrombin (200 nM)increases PLA, activity ) about 20-fold if PI is used as a substrate,about 50-fold if PE is the substrate, and about 100-fold for PC as thesubstrate (FIG. 8). The activity is resistant to reducing agents and,therefore, not a secreted PLA₂ (FIG. 8B). Because of the substrateselectivity, the cPLA₂ is not cPLA₂-85 but a different enzyme or acombination of different enzymes similar to those discovered in growthcones (Negre-Aminou et al., 1996). Recombinant SemaIV, the putativetumor suppressor described in Roche et al., Oncogene 12: 1289-1297(1996), also increases cPLA₂-PE activity several fold, in adose-dependent manner, and also causes pseudopod retraction in MAT-Lucells. Therefore, thrombin and SemaIV stimulate cPLA₂ and pseudopoddetachment/retraction in these cancer cells.

Experiments were conducted to determine the effects of free AA and itsmetabolites (eicosanoids) on MAT-Lu pseudopods. Because these agents areunstable, pseudopod length measurements were performed after shortincubations, rather than Boyden chamber assays. Typically, cells wereexposed to AA or eicosanoid (with or without inhibitor) for 15 min andthen digitally imaged (see above). Pseudopod lengths were measured andtheir distribution (in μm) subjected to population analysis (expressed,e.g., as quantiles in Whisker-Box plots). Micromolar AA (but not stearicacid used as a control) caused significant but reversible reduction ofpseudopod length (FIG. 11A). The cyclooxygenase inhibitor, indomethacin,did not influence the AA effect, but the lipoxygenase inhibitor,nordihydroguaiaretic acid (NDGA), and the more selective 12-LOX blocker,cinnamyl-3,4-dihdroxy-α-cyanocinnamate (CDC) described in Cho et al., JMed Chem 34: 1503-1505 (1991), blocked the AA effect completely (FIG.11B). In fact, at the IC₅₀ or above, CDC seemed to cause pseudopodelongation. This pointed to an important role of 12-LOX and 12(S)-HETEin the observed phenomenon.

Results from Western blots of cultured MAT-Lu cells are consistent withthis conclusion. Cultured MAT-Lu cells were harvested, solubilized andthe proteins resolved by SDS polyacrylamide gel electrophoresis. Blotsof these polypeptides were probed with an antibody to leukocyte 12-LOX,provided by Dr. C. Funk, University of Pennsylvania. The blots revealeda single, prominent band of the appropriate M_(r) (75,000 Da) indicatingthe presence of this enzyme.

The effects of 12(S)-HETE on MAT-Lu pseudopods are shown in FIGS. 16 and18. The length of these pseudopods is sensitive to 12(S)-HETE. Thedose-response is biphasic with a peak of pseudopod detachment andretraction between 10⁻¹⁰ and 10⁻¹¹M. This phenomenon mirrors the MARCKSphosphorylation response observed in isolated growth cones (FIG. 7B).The isomer 15(S)-HETE was about as active as 12(S)-HETE, but 12(R)-HETE,the stereoisomer of 12(S)-HETE, and 5(S)-HETE were essentially inactivebetween 10⁻¹⁰ and 10⁻⁶ (FIG. 12). The effective 12(S)-HETEconcentrations and the time necessary to see a response in ourexperiments were well below the K_(d) (10⁻⁹ M) and the saturation time(90-120 min) for the putative 12(S)-HETE receptor reported byHerbertsson and Hammarstrom, FEBS Lett 298:249-252 (1992) and Liu et al.(1995). Except for two papers which describe effects of 10⁻¹⁰ to 10⁻⁸12(S)-HETE on neutrophil motility (Goetzl et al., J clin Invest59:179-183 (1977) and Yoshino ct al., Gen Pharmac 24:1249-1251 (1993),most or all published studies with this eicosanoid have been performedat much higher concentrations so that the effects reported here were notobserved. The 12(S)-HETE effect is PKC-dependent. Preincubation ofcultured cells with the PKC inhibitor, calphostin, not only blockspseudopod retraction normally caused by 10⁻¹⁰ 12(S)-HETE, it seems toresult in pseudopod elongation (FIG. 12D), as determined in thepseudopod length assay.

These observations support that thrombin-elicited pseudopod detachmentand retraction of a highly motile cancer cell line are mediated b cPLA₂,eicosanoid production, activation of PKC, and phosphorylation of MARCKS(see FIG. 2).

EXAMPLE 4 The Signaling Cascade and its Modulation in Other Cancer CellLines

The cascade is compared in detail in four Dunning carcinoma cell linesof different motility: AT-2 (low) versus AT-3. MAT-LyLu and MAT-Lu(high). Based on their behavior and growth pattern in culture and, whereavailable, on the results of Boyden chamber assays, AT-2 cells are muchless motile than the other cells. Thrombin stimulated cPLA₂ 40-50-foldin MAT-Lu and about 30-fold in AT-2 cells (FIGS. 8 and 9), when PE wasthe substrate. For the substrates PI and PC the stimulation of cPLA₂ waseven greater in AT-2 than in MAT-Lu cells, over 100-fold. This indicatedthat AT-2 cells contain the thrombin receptor, the signaling mechanismregulating cPLA₂, and the appropriate forms of activatable cPLA₂.However, AT-2 cells essentially did not respond to thrombin in thepseudopod retraction assay. Furthermore, AT-2 cells were almostcompletely insensitive to AA, even at 10⁻¹M, whereas all threehigh-motility cell types responded to AA in the manner described forMAT-Lu cells. Regarding 12-LOX, AT-2 and MAT-Lu cells exhibited similarlevels of enzyme protein, as determined by Western blot. The pseudopodresponses to 12(S)-HETE were of particular interest. As shown in FIG.16, high-motility cells showed various degrees of pseudopod retractionpeaking at 10⁻¹¹ to 10⁻¹⁰M 12(S)-HETE, whereas retraction was notobserved in AT-2 (FIGS. 13 and 16). 10⁻¹¹M 12(S)-HETE seems to increasepseudopod length in AT-2 cells in the pseudopod length assay. Theseobservations indicated that AT-2 cells are deficient in the signalingpathway leading to pseudopod withdrawal, and that the defect must bedistal to the generation of 12(S)-HETE.

Various human prostatic carcinoma (CaP) lines were obtained from Dr. G.Miller. University of Colorado, Denver Colo. (LNCAP, PPC-1, PC3. DU145,TSU-Pr1) and Dr. I. Fidler, M. D. Anderscn, Houston Tex., (PC-3M andPC-3M-MM2) (Table 2). Experiments were designed to determine thevalidity of some basic observations in these human CaP cells.

The motility of PC-3M-MM2 cells was examined in Boyden chamber assaysand thrombin was found to be a potent inhibitor of cell motility (Table1). The human CaP cells were tested for their pseudopod responses tothrombin and 12(S)-HETE. As indicated qualitatively in Table 2, allcells tested retracted pseudopods in the presence of the two reagents,albeit to different degrees. Stimulation of cPLA₂ activity by thrombinwas measured in PC-3M-MM2 cells. Resting AA release from PE and PC wasfound to be quite low, but it was stimulated about 4-fold by thrombin.In contrast, resting levels for PI hydrolysis were much higher but notfurther stimulated by thrombin. PC-3M-MM2 cells contain about the same,high level of 75-kDa leukocyte-type 12-LOX observed in the other cellstested as determined by Western blot of PC-3M-MM2 polypeptides resolvedby gel electrophoresis.

TABLE 2 Human CaP Cell Lines Estimated Pseudopod retraction* metastatic12(S) Type Source potential References thrombin HETE PC-3 Bone meta. ofprostate Low Kaighn et al., 1979 + + PC-3M Sfrmpvs/ Moderate Koslowskiet al. 1984 + + PC-3M-MM2 Liver meta. of PC-3 High Pettaway et al.,1996 + + 2-3 rounds of meta. selection after PC-3M injection LNCaP Lymphmeta. of Low-moderate Horoszewixz et al. 1983 + + prostate adenoca. DU145 Brain meta. of prostate Moderate Stone et al. 1978 + + adenoca.PPC-1 Primary prost. Low Brothman et al., 1989 nd nd Adenoca. TSU-Pr1Lymph meta. of Low-medium Iizumi et al., 1987 + + prostate adenoca.*Microscopic examination: nd, not done

These experiments demonstrate: (i) the proposed cascade is operationalin various motile cancer cells, including human CaP cells, and (ii) itappears differentially modulated in cancer cells of varying motility.

In addition, these results and the data summarized above support theconcepts that: (i) growth cone and CaP cell motility is regulated notonly by positive-chemotactic factors but also by repellent factors, suchas thrombin, SemaIV and netrin; (ii) their inhibition of motility andpseudopod repulsion is mediated by the proposed cascade, and (iii) thesignaling pathway seems to change with motility levels and metastaticprogression of cancer cells.

The following examples demonstrate that the pseudopod repellentmolecules, thrombin and SemaIV, and probably Semaphorin III and netrin,stimulate the signaling cascade and trigger adhesion site disassembly inhuman CaP cells.

The data demonstrate the existence (inside as well as outside thenervous system) of a mechanism that controls cancer cell motility andneurite growth via the action of pseudopod repellent factors. Thefollowing examples set forth the proposed mechanism of action of thesefactors. Data indicate a critical role for the cPLA₂-cicosanoid-PKCcascade, and they suggest adhesion site disassembly as its target. Thisindicates that pseudopod repellents can act to suppress tumor cellmotility and dissemination.

EXAMPLE 5 Analysis of the Repellent-activated Signaling Pathway

The human CaP cell line(s) are analyzed systematically according to theschematic shown in FIG. 2. Repellent activation of cPLA₂ (usingdifferent substrates, PE, PC or PI), 12-LOX and PKC, in the presence orabsence of an inhibitor of the step just upstream of the enzyme ofinterest, are measured. This gives a detailed view of the biochemical,structural and behavioral responses of CaP cells to repellent factors.

PLA₂ stimulation. Using ¹⁴C-AA-PE, -PC or -PI as a substrate, thrombin,SemaIV and netrin activation of the enzyme are measured. For theseassays, commercially available thrombin or thrombin-receptor-activatingpeptide (TRAP) (Sigma, St. Louis, Mo.), recombinant SemaIV provided tous by Dr. H. Drabkin, University of Colorado School of Medicine,recombinant SemaIII provided by Dr. Y. Luo, Exelixis Pharmaceuticals,San Francisco Calif., and recombinant netrin from Dr. M.Tessier-Lavigne, (University of California, San Francisco) are used.Time courses and dose-response curves are generated with the appropriatesubstrate(s). These assays are carried out according to well-establishedprotocols described in Negre-Aminou et al., 1996, supra, incorporatedherein by reference, on cells grown to equal density levels (asdetermined by protein and DNA content). Some enzyme assays are carriedout in the presence of reducing agent, which blocks secreted but notcytosolic PLA₂ activity, to exclude potential interference by secretedPLA₂. To characterize the stimulated cPLA₂ further, cultured cells arehomogenized in protease inhibitor-containing buffer, in the presence of1 mM EDTA or 0.3 mM Ca²⁺, and then centrifuged in order to generatecrude membrane fractions and cytosolic extracts. Both of these areanalyzed (at equal calcium levels) for cPLA₂ activity to determinewhether CaP cPLA₂ associates with the membrane in a calcium-dependentmanner (as seen for cPLA₂-85). In separate experiments, enzyme activityis measured in the presence of different concentrations of free calciumto determine whether the enzyme activity is calcium-dependent or not

12-LOX protein and activation. The presence of leukocyte-type 12-LOX inprostate carcinoma (CaP) cells is determined by Western blot, usingantibody received from Dr. Colin Funk. University of Pennsylvania. CaPprotein is run alongside a standard sample of rat peritonealmacrophages. 12-LOX activity is assayed in two different ways, with¹⁴C-AA or with ¹⁴C-AA-PE (or -PI or -PC) as substrate. In both cases,the generation of ¹⁴C-labeled 12(S)-HETE is measured, but in the lattercase the measured value is a composite of PLA₂ and 12-LOX activity.Comparison of the results indicates whether 12-LOX is regulated orconstitutively active. After incubation of homogenates ofrepellent-pre-treated or control cells with the substrate, labeledproducts are extracted with chloroform according to Salmon and Flower(1982). The extracts are dried and run on thin-layer plates, togetherwith 12(S)-HETE standards. The 12(S)-HETE bands are scraped off andcounted in a scintillation spectrometer. Selected samples are processedby “solid phase extraction” on Sep-Pak columns, followed by HPLC asdescribed in Yu and Powell, Analy Biochem 226:241-251 (1995),incorporated herein by reference, and counting of the fractions.Absorption peaks are monitored at 237 nm, the maximum absorption ofHETEs using an ultraviolet detector with variable wavelength for theHPLC instrument. Alternatively, extracted HETE can be identified andquantified mass spectrometrically. The results are expressed as pmoleslipid messenger synthesized per minute, per mg protein. In experimentsinvolving ¹⁴C-AA-PE (or -PI or -PC) as a substrate, cPLA₂ inhibitorsalso are used. These are: bromoenol lactone (Ackermann et al., J BiolChem 270:445-450 (1995)) or the AA analog AACOCF₃ (Street et al.,Biochem 32:5935-5940 (1993), one of which should block therepellent-stimulated generation of ¹⁴C-AA and of ¹⁴C-12(S)-HETE (theblocker, p-bromo-phenacylbromide, is too non-specific). Theseexperiments indicate (i) that stimulation of CaP cells by the differentrepellents increases the generation of 12(S)-HETE, as shown for MAT-Lucells and growth cones and thrombin, and (ii) that this is due to theincreased availability of AA and/or increased 12-LOX activity itself.The data generated herein, as well as those in the literature (Shimizuand Wolfe, J. Neurochem 55:1-15 (1990)) suggest that generation of AA isthe rate-limiting step in eicosanoid production and that 12-LOX isconstitutively active rather than being activated by the signalingpathway.

PKC activation. Repellent stimulation of CaP cells increasephosphorylation of endogenous MARCKS or an exogenous substrate, MARCKSphosphorylation site domain (PSD) peptide (commercially available fromCalbiochem, LaJolla, Calif.) was determined. CaP cells in culture arepre-loaded with ³²P-orthophosphate and stimulated with differentrepellent concentrations for different time intervals (0.5 to 10 min).The reaction is stopped and MARCKS extracted with ice-cold acetic acidas described in Robinson et al. Analyt Biochem 210:172-178 (1993).Extracted polypeptides are run on SDS-polyacrylamide gels (SDS-PAGE). AMARCKS antibody is used for positive MARCKS identification by Westernblot and/or for immunoprecipitation. Attentively, two-dimensional gels,in which MARCKS forms a characteristic triangular and highly acidic spotas described, for example, in Katz et al., J Neurosci 5:1402-1411 (1985)can also be used. Phosphopeptide maps of this spot also are generated toascertain positive identification (Wu et al., PNAS, USA 79:5249-5253(1982): Katz et al., 1985). MARCKS phosphorylation is determinedquantitatively in the phosphorimager and expressed based on total cellprotein in the assay. Alternatively, CaP cell homogenates are incubatedwith ³²P-ATP in the presence or absence of repellent. Phosphorylationsubstrate is endogenous MARCKS or exogenous PSD peptide. The reaction isstopped with ice-cold acid, and the supernatant containing theacid-soluble MARCKS or PSD is analyzed by gel electrophoresis and thephosphorimager. The results indicate (i) whether, (ii) with which timedelay and (iii) at which concentration each repellent stimulates PKCactivity as determined by MARCKS or PSD phosphorylation. Based on thedata obtained on isolated growth cones, a dose-dependent phosphorylationof MARCKS in response to repellent incubation should be found.

These experiments provide quantitative data on the activation of thevarious steps of the cascade by each of the repellents tested. Theinhibitor experiments confirm the dependence of later steps in thecascade on the proposed preceding steps. These biochemical data arecorrelated with the observations on motility and adhesion sitemorphology from Example 2.

EXAMPLE 6 Phosphorylation and Adhesion Site Disassembly

This example demonstrates that phosphorylation of MARCKS and itssubsequent dissociation from adhesion sites triggers the disassembly ofthe adhesion site. The analysis of such mechanisms cannot be performedin whole cells. Instead, adhesion site preparations are generated byplating CaP cells on laminin, collagen, fibronectin or other similarsubstrata prepared by coating culture dishes first with nitrocelluloseand then with the substrate protein as described in Lagenaur and Lemmon,PNAS, USA 84:7758-7757 (1987). Such substrata are resistant todetergents including SDS (important for the processing of the samples).After several hours of growth on these substrata at low density, CaPcells are extracted with Triton X-100 (TX100) in order to remove allmembrane and soluble proteins that are not attached to the substratum orthe cytoskeleton. This procedure leaves adhesion sites intact forbiochemical or morphological analysis as described in Rohrschneider,PNAS, USA 77:3514-3518 (1980) and Sobue & Kanda, Neuron 3:311-319(1989). The biochemical analyses are paired with immunofluorescencelocalization studies. Adhesion site disassembly is monitored by therelease of actin, characteristic adapter proteins (for example, talin,paxillin and vinculin), and integrin with probes that arc commerciallyavailable.

The first set of experiments is designed to examine PKC activation by12(S)-HETE. From preliminary studies on growth cones, we know that theTX100-extracted adhesion site preparation retains PKC activity andMARCKS. Because MARCKS availability may be limiting, exogenous PSDpeptide is used as a substrate. The adhesion site preparations arewashed with buffer and then incubated for different periods with PSD,12(S)-HETE (or 12(R)-HETE as control) and ³²P-ATP at different, lowcalcium levels. In parallel experiments, diacylglycerol and AA are usedas potential PKC activators, again at different calcium levels. After 10sec to 1 min at 30° C., the reactions are stopped. The solublesupernatant of the preparation is collected and analyzed by SDS-PAGE andphosphorimaging. Time courses and dose response curves of PSDphosphorylation are analyzed to determine the profile of PKC activation.

12(S)-HETE-stimulated phosphorylation of endogenous MARCKS and itsdissociation from adhesion sites also is studied. Adhesion sitepreparations are incubated as described, but without PSD peptide. Thesoluble supernatant is collected. The proteins that remain attached tothe substratum also are solubilized with SDS. Phosphorylation of MARCKSis determined in both protein preparations. MARCKS is acid-extracted andthen run on one- or two-dimensional gels. Alternatively, MARCKS may beimmunoprecipitated with antibody, using radio-immunoprecipitation assay(RIPA) buffer (containing TX100, deoxycholate and SDS). Once MARCKS hasbeen identified, further protein analysis is done by SDS-PAGE, if thereare no other phosphoproteins seen at that Mw. One or several of theseprocedures are used to identify MARCKS in the substrate-bound and thesupernatant fractions, and its phosphorylation is quantified byphosphorimaging. The data obtained are expressed as ³²p incorporationinto MARCKS of each fraction, as a function of 12(S)-HETE concentration.Most phospho-MARCKS is found in the supernatant. Using MARCKS antibodyin Western blots allows one to quantify, in the same phosphorylationexperiments, the relative distribution of MARCKS protein between thesubstrate-adherent and the soluble fractions. Western blots of the twofractions are probed with the MARCKS antibody and then an HRP-conjugatedsecond antibody. After visualization of MARCKS, semi-quantitative datacan be obtained by densitometry of the blot.

PKC-activation triggering adhesion site disassembly also is examined asindicated by the loss of characteristic adhesion site proteins into thesoluble supernatant. PKC in adhesion sites is stimulated (or controlincubated), in the presence of unlabeled ATP, with or without an optimaldose of 12(S)-HETE (substitution of 12(R)-HETE or addition of the PKCinhibitor calphostin serve as controls). Substrate-bound and solublefractions are prepared and processed for Western blot analysis. Theblots are probed with antibodies to talin, paxillin and vinculin, whichare key components of adhesion sites. Using the semi-quantitativeapproach described above, the relative amounts of talin, paxillin andvinculin are monitored in the two fractions as a function of (i)12(S)-HETE concentration in the assay and (ii) incubation time (up to 5min). Because these experiments are carried out on laminin, collagen,fibronectin or similar substrata) matrices, we predict the involvementof particular integrin subunits (most likely β₁, but possibly β₄ forlaminin, Albelda, Lab Inves. 68;4-17 (1993)). This is verified byprobing first CaP cell membrane preparations and then adhesion sitepreparations for the presence of integrin β₁ and β₄ by Western blot(antibodies available commercially from Chemicon. Temecula, Calif. orLife Technologies/GIBCO-BRL, Gaithersburg, Md.). If PKC-activationtriggers adhesion site disassembly, integrins disaggregate anddissociate from their ligands. Therefore, 12(S)-HETE-treated (vs.control) adherent and soluble fractions are examined by Western blot forthe presence of integrins. In further experiments, adherent proteinsfrom radio-phosphorylation experiments are solubilized in RIPA buffer,followed by immunoprecipitation of the integrin from this fraction aswell as from the soluble supernatant. The immunoprecipitates areresolved by SDS-PAGE and analyzed with the phosphorimager to determinewhether PKC activation with 12(S)-HETE also phosphorylates the integrinβ subunit, and whether this correlates with integrin distribution,between the attached fraction versus the Triton X-100 solublesupernatant.

It is helpful to pair the biochemical experiments with morphologicalanalyses. CaP cells are grown on coverslips coated with laminin,collagen, fibronectin or other similar substrata, subjected for variousperiods of time to treatment with repellent or 12(S)-HETE and then fixedmildly with a low concentration of formaldehyde. Subsequently cells arepermeabilized with TX100 (at a low concentration in order to preservemost of the membranes) to allow for the escape of soluble proteins. Suchpreparations are quenched with glycine and bovine serum albumin and thenprocessed for immunofluorescence with primary antibody, followed by atagged secondary antibody. Controls consist of omitting the primaryantibody and using instead non-immune serum from the same species. Inaddition to the antibodies to talin, paxillin, vinculin and integrin β₁(or β₄), MARCKS antibody and fluorescent phalloidin (to labelpolymerized actin) also are used. Preferably samples are examined byconfocal microscopy. When advisable, double-immunofluorescence withphalloidin and differently tagged secondary antibodies are performed.The presence of MARCKS, talin, paxillin, vinculin, actin and theappropriate integrin are studied morphologically in pseudopod attachmentsites in control cells and during their changes in response torepellents or 12(S)HETE treatment.

This example provides information about the molecular rearrangementsoccurring in adhesion sites in response to repellent treatment. As ingrowth cones, adhesion site PKC in CaP cells also is expected to respondrapidly and in a biphasic manner to very low concentrations of12(S)-HETE. However, such a response would not exclude intermediatesteps between 12(S)-HETE release and PKC activation. A biphasic doseresponse of PKC to 12(S)-HETE is important for understanding how aparticular balance of MARCKS phosphorylation is maintained in the cellduring locomotion. The example also demonstrates that phospho-MARCKSdissociates from the adhesion sites, and that this is correlated with,or triggers, the disassembly of the entire adhesion site, includingintegrin detachment. There may be other substrates of PKC that areimportant in the phenomenon.

Experiments also arc performed to determine whether phospho-MARCKSdissociates from the adhesion sites and whether this is correlated with,or triggers, the disassembly of the entire adhesion site, includingintegrin detachment.

EXAMPLE 7 Thrombin as a Pseudopod Repellent

In this example, a highly motile cancer cell line with long, almostneurite-like processes, the MAT-Lu subline of Dunning rat prostaticcarcinoma was used. We found that thrombin acts as a pseudopod repellentin these cells, and that it is a strong activator of cPLA₂. A variety ofinhibitor experiments indicate that the generation of 12(S)-HETE as wellas the activation of protein kinase C (PKC) are necessary for thrombin'srepellent effect. The application of intermediates of the signalingpathway, such as AA or 12(S)-HETE, to the cells indicate that thesereagents are sufficient to cause pseudopod detachment and retraction.Therefore, the present example relates to the signaling pathway thatlinks pseudopod detachment and retraction to thrombin receptoractivation.

Cell Culture. MAT-Lu cells were grown in RPMI 1640 cell culture medium(e.g., Sigma) containing 10% heat-inactivated fetal bovine serum, 2 mML-glutamine, 100U/ml each of penicillin and streptomycin, and 250 nMdexamethasone, in 5% CO₂ in air at 37°, as described by Isaacs et al.(1986). Cells were monitored morphologically to ascertain the constancyof the phenotype.

Boyden Chamber Motility Assays. Disposable transwells (Coming Costar,Cambridge, Mass.) of 6.5 mm diameter and 8 μm pore size were coated with5 μg/ml laminin on both sides. The membranes were washed twice withHank's balanced salt solution (HBSS) and then pre-incubated with growthmedium containing 10% fetal bovine serum. Trypsinized cells (5×10⁴) wereplated into the upper well. After 1 hour the upper chamber was washedtwice with HBSS, filled with serum-free medium and moved to a new lowerchamber, also filled with serum-free medium, with or without thrombinand/or 0.75 nM insulin-like growth factor-1 (IGF-1).

After incubation for 4 hrs, cells on the membrane were fixed and stainedwith a Diff-Quick kit (Baxter Scientific Products). Cells on the upperside were scraped off, the membranes were removed, mounted ontomicroscope slides and analyzed. Each filter was scanned twice at rightangles under the light microscope with a 16×phase contrast objectivelens. A continuous series of 20 frames for each filter was digitizedusing a high-resolution CCD camera (Kodak) and stored in a PowerMacintosh computer. The two most peripheral pairs of frames from eachscan were deleted so that 12 frames were analyzed for each filter. Cellsthat had crossed the membrane were counted and tabulated. The sum of thecells counted in the twelve frames was an individual data point.

Pseudopod Morphometry. To study the effects of various agents onpseudopods on a short-term basis, we measured pseudopod lengths. Cellsplated in 35-mm diameter dishes at low density (1×10⁴ per dish) wereincubated overnight, tie medium was changed, and the experiments wererun four hours later. First, cultures were shifted to serum-free medium,which was replaced one hour later by serum-free medium with or withoutthe various agents (thrombin, AA or HETEs). Care was taken to avoid heatand light inactivation of the eicosanoids. Culture dishes were placed ona Zeiss inverted microscope equipped with a heated stage and viewed witha 16×phase contrast objective lens. Randomly selected fields of thecultures, containing generally about 20 cells each, were recorded,digitized and stored in the computer at the onset of the experiment andat different times thereafter.

In some cases, individual cells were monitored over different periods oftime immediately after introduction of the reagent, either by mixing itinto the medium or by applying it locally to one end of the cell. Localapplication was done with a “puffer” system that expels microliteramounts of the desired solution from a micromanipulator-controlledmicropipette tip. Cell morphology was recorded, and the captured imageswere analyzed with a Power Macintosh computer using NIH Image. Cellprocess length was measured from the center of the nucleus to the end ofthe process. Characterization of the frequencies of pseudopod lengths,at a fixed time for each of a sequence of concentrations of reagent,utilized a gamma regression model with an inverse quadratic linearpredictor (McCullagh and Nelder, Generalized Linear Models 2d ed., pp.291-292 (Chapman & Hall, 1989), separately fit to each concentrationdata set. Although no formal goodness-of-fit tests were carried out, invirtually every case there was good visual representation of the patternof response (see FIG. 14). Most data, however, are shown as whisker-boxplots, where the center point represents the 50%, the ends of the boxthe 23% and 75%, and the “whiskers” the 5% and 95% quantiles (e.g., FIG.11). Statistical comparisons of pseudopod lengths for differenttreatments were done by standard non-parametric techniques (WilcoxonRank Sum or Kruskal-Wallis Tests), as judged appropriate. Polynomialcurve fittings were done by standard least-squares techniques.Statistical analyses and graphing were done with the use of SASprocedures: Reg, Phreg, Nparlway and Plot (SAS institute Inc., Cary,N.C.).

Biochemical Assays. MAT-Lu cells were plated at a density of 0.5×10⁶cells per flask (T-75) and grown as detailed above to approximately 25%confluency. On the day before the experiments, the serum level in themedium was dropped to 1%. Cells were fed with fresh 1% serum-medium 4hrs prior to harvest. For harvesting, cells were rinsed withCa⁺⁺,Mg⁺⁺-free HBSS, covered with 2 ml cold Ca⁺⁺-free modified Krebsbuffer (220 mM sucrose, 50 mM NaCl, 5 mM KCl, 22 mM HEPES, 10 mMglucose. 1.2 mM NaH₂PO₄ and 1.2 mM MgCl₂, pH 7.3), and placed on ice.Cells were scraped off, pelleted for 5 min at 200 rpm and re-suspendedin a minimal volume of Krebs buffer. Aliquots were used to determineprotein concentration (Biorad Assay) and phopholipase activity. Tomeasure PLA₂ activity 10-12 μg of cell protein was incubated with orwithout thrombin for 10 min on ice, ¹⁴C-AA-labeled phospholipidsubstrate (7 μM. 40-60 mCi/mmol) was added, and the mixture wastransferred to 37° for 20 min. The reaction was stopped by adding coldcliloroform/methanol (1:2 v/v). Lipid extraction followed by thin-layerchromatography (Negre-Aminou et al., J Neurochem 67:2599-2608 (1996))isolated the reaction product (identified by co-migration with AAstandard), which was then scraped off the plates and analyzed in ascintillation spectrometer (Beckman). In some experiments, especiallythose involving ¹⁴C-AA-phosphatidylinositol as substrate, thediacylglycerol-containing band (identified by co-migration of standard)also was scraped and counted in order to measure phospholipase C (PLC)activity.

The generation of 12(S)-HETE in MAT-Lu cells in response to thrombin wasassayed in cells cultured and harvested as just described. Ten to twentymg cell protein was incubated with or without thrombin for 10 min onice, ¹⁴C-AA-labeled phospholipid or ¹⁴C-AA was added as substrate, andthe mixture was transferred to 37° for 10 or 20 min. The reaction wasstopped by the addition of ice-cold chloroform. Lipid extractionfollowed by thin-layer chromatography or HPLC isolated the reactionproduct [12(S)-HETE], identified by co-migration of standard.

Cell Motility. MAT-Lu cells arc highly motile (Isaacs et al., 1986).They immediately spread over the culture dish rather than forming clonalcolonies. Boyden chamber assays quantify motile behavior by measuringthe number of cells that migrate through the pores of a membrane withina specific period of time (Boyden, 1962). The laminin-coated filters inour assays had relatively large, open pores (8 μm) so that proteolysisof extracellular matrix was not necessary for crossing the membrane.IGF-1, which is a positive chemotactic factor for many cell typesincreases MAT-Lu migration by about 80% in four-hour assays (Table 1).Thrombin, in contrast, inhibits both control and IGF-1-induced migrationacross the membrane in a dose-dependent manner (FIG. 10), resulting in a70-80% decrease (at 200 nM). Thus, thrombin acts as a negativechemotactic factor for MAT-Lu cells.

Because of the involvement of HETEs in the mechanism of thrombin action,Boyden chamber assays also were performed with thrombin in the presenceof the LOX inhibitor, cinnamyl-3, 4-dihydroxy-alpha-cyanocinnamate(CDC). At 63 nM (IC₅₀) and at 126 nM CDC inhibits 12-LOX selectively(Cho et al., 1991). Table 1 shows that CDC reverses the effect ofthrombin.

Effects of Thrombin and Eicosanoids on Pseudopod Length. Boyden chamberassays cannot produce short-term data on motility mechanisms. Therefore,morphometric assays were used to observe cellular effects immediatelyafter addition of a reagent. In these assays, thrombin (200 nM) causedpseudopod shortening in MAT-Lu cells within minutes of administration.Typically, the cell processes are seen to retract slowly, withpersisting knob-like enlargements at their tips. The withdrawingpseudopods frequently are curvilinear, indicating that they are notunder tension. These morphologies suggest that distal pseudopoddetachment is the primary event of thrombin-induced process retraction.The phenomenon of pseudopod withdrawal is reversible after thrombinremoval.

The effects of AA, a potential mediator of thrombin action, also wereinvestigated. Exogenous AA in the micromolar range (but not other fattyacids, such as stearic and linoleic acid) mimics the thrombin-inducedlength reduction within a similar time frame (FIG. 15A; 15 min). Alinear regression of response data points across the logs of AA dosesyielded a significant (p<0.002) slope of −1.26.

A 15-min pretreatment with the cyclo-oxygenase inhibitor, indomethacin(10 ηm), had no effect at all on AA-induced pseudopod shortening.However, nordihydroguiaretic acid (NDGA), which blocks LOXsnon-selectively at 25 μM, and CDC did interfere with the AA effect. Byitself the less specific NDGA (25 μM; 15 min pretreatment) had no effecton pseudopod lengths, but it inhibited the pseudopod shortening observedat 10⁻¹ M AA so that control and AA+NDGA samples were not significantlydifferent (p>0.75). FIG. 15B shows the results obtained with CDC, whichis selective for 12-LOX. At 63 nM (IC₅₀) and at 126 nM, CDC pretreatment(15 min) reversed the AA effect so that controls and AA+CDC samples wereindistinguishable statistically (whereas the values for AA alone weresignificantly lower, at p<0.01). These results suggest the involvementof 12-LOX in the retraction phenomenon. Therefore, subsequentexperiments were focused on the 12-LOX product of AA, 12(S)-HETE.

The general or localized application of 12(S)-HETE to MAT-Lu cells inculture was examined. Microliter amounts of 12(S)-HETE (10⁻¹⁰M) releasedinto the medium about 10 μm away from a particular cell process,resulted in the onset of pseudopod detachment and shortening within 1min. After about 15 min, the pseudopod became absorbed completely intothe cell, and several times the emergence of a new pseudopod wasobserved on the opposite pole. If 12(S)-HETE (10⁻¹⁰M) was applied bymedium replacement, pseudopod shortening was nearly complete for mostcells within about 15 min. The observed morphologies were the same asthose for thrombin. Thirty minutes after withdrawal of 12(S)-HETE bymedium replacement (done 30 min. after onset of the experiment),pseudopods had reappeared indicating reversibility of the eicosanoideffect.

Dose responses of pseudopod lengths to different HETEs were measuredafter 15 min incubation. Raw data for 12(S)-HETE are shown in FIG. 14.As expected, pseudopod lengths did not form a Gaussian distribution.However, the data could be fitted with a gamma regression model. Thevertical lines in the histograms indicate the mode. It shifts to ashorter pseudopod length at 10⁻¹¹ and 10⁻¹⁰ M and then back toapproximately control levels at higher concentrations of 12(S)-HETE. Thewhisker-box plot in FIG. 12A shows more clearly the change in pseudopodlength distributions induced by 12(S)-HETE. At 10⁻¹¹ and 10⁻¹⁰ M, valuesare significantly smaller than control (>45% length reduction for themedian; p<0.0001). The overall curvilinear depressed pattern, withpseudopod lengths returning to control levels at the higherconcentrations (10⁻⁸ and 10⁻⁶M), could be fitted with a quadraticpolynomial across the log doses −12 through −8 (p<0.0001). This curvedisplayed a minimum at log −10.4M. The time course of pseudopodshortening is shown in FIG. 15 for a population of 73 identifiedpseudopods upon general application of 10⁻¹⁰ M 12(S)-HETE. As a controlfor 12(S)-HETE, we used its stereoisomer in the same experiments. At10⁻¹⁰, but not at 10⁻⁶ M 12(R)-HETE, we observed a small reduction inpseudopod length (FIG. 12B). Two other isomers of 12(S)-HETE were testedas well. 5(S)-HETE at 10⁻¹⁰ M had no effect on pseudopod length (FIG.12B). However, 15(S)-HETE reduced pseudopod lengths of MAT-Lu cells in abiphasic manner, like 12(S)-HETE (FIG. 12C). The effect was significant(p<0.0001) and peaked at log −10.0M. In summary, 12(S)-HETE and15(S)-HETE caused a rapid and dramatic, biphasic reduction in pseudopodlength, with the maximum effect observed between 10⁻¹⁰ and 10⁻¹¹ M,whereas the other isomers tested were essentially inactive.

Further downstream of eicosanoid generation, 12(S)-HETE may directly orindirectly activate protein kinase C. To test the role of PKC inpseudopod detachment and shortening the blocker calphostin C was used topretreat cells for 15 mins. (Kobayashi et al., Biochem. Biophysi. Res.Comm. 159:548-553 (1989)) in conjunction with 12(S)-HETE (10⁻¹⁰M). Asshown in FIG. 12D, at 50 nM (IC₅₀) and at 100 nM, calphostin completelyinhibited pseudopod withdrawal triggered by 12(S)-HETE (p<0.0001).

Phospholipase Activity in MAT-Lu Cells. The behavioral and morphometricstudies suggest that thrombin stimulates cPLA₂ in MAT-Lu cells.Therefore, AA release from exogenous ¹⁴C-AA-phosphatidylethanolamine(PE), -phosphatidylcholine (PC) and phosphatidylinositol (PI) weremeasured. Although AA release from PI is higher than from the othersubstrates under resting conditions, stimulation with thrombin resultsin a large increase (for PC>100 fold) in AA release, to about the samemolar levels for all three substrates (FIG. 8A and B). Under the sameconditions of thrombin stimulation, we also looked for changes in¹⁴C-AA-diacylglycerol (DAG) release from ¹⁴C-AA-PI (FIG. 8A). While DAGrelease from PI was much higher than that of AA under restingconditions, thrombin actually reduced DAG release rather thanstimulating it. There was essentially no DAG release from PE and PC. Totest for the possible involvement of secreted PLA₂ we performed PLA₂assays in the presence of reducing agent (5 mM dithiothreitol, DTT),which inhibits secreted PLA₂s but leaves cytosolic PLA₂s unaffected. Asshown in FIG. 8B, DTT did not significantly inhibit the measured PLA₂activity. The dose response of cPLA₂ to thrombin is shown in FIG. 8C forthe PE substrate.

Thrombin Effects on MAT-Lu Cell Behavior. Thrombin greatly inhibitedcell motility in our Boyden chamber assays and, thus, has a negativechemotactic effect on the MAT-Lu cells. Short-term morphometric studiesrevealed that pseudopod withdrawal was the reason for decreased cellmigration. Therefore, thrombin's effect on these cells is that of arepellent, similar to what can be observed with growing neurites(Monard, Trends Neurosci 11:541-544 (1988)). Morphological observationssuggest that the pseudopods react to thrombin and 12(S)-HETE first bydetachment, rather than by increased tension and subsequent passivedisruption of attachment sites. Therefore, thrombin and eicosanoidsexert their effects on cellular adhesion sites.

Thrombin Activation of PLA₂. Analysis of reaction products fromradiolabeled-PI substrate included not only AA but also DAG. Thrombindid not increase the levels of radiolabeled-DAG compared to controlsand, therefore, did not activate phospholipase C in MAT-Lu cells. Incontrast, AA release from PI was stimulated about 18 fold by thrombin,and it was increased 36 or 113 fold, respectively, when PE or PC wereused as substrates. However, the molar levels of AA released from theseexogenous substrates at maximum thrombin stimulation were about thesame. Resistance to reducing agent and activity at low calcium levels(100 μM) indicate involvement of cPLA₂. Several forms of cPLA₂ have beencharacterized to date (Dennis, J Biol Chem 269:13057-13060 (1994);Leslie, J Biol Chem 272:16709-16712 (1997)). The substrate selectivityobserved is consistent with the activation of a PI-selective enzymepreviously observed in nerve growth cones (Negre-Aminou et al., 1996),perhaps together with cPLA₂-85, the PE-and PC-selective 85-kDa enzyme.

If cPLA₂ is causally involved in pseudopod withdrawal, then itsinhibition ought to block the effect of thrombin. Likewise, if cPLA₂ isinvolved in this pathway, then one of the enzyme's products, AA or alysophospholipid, should have the same effect. Micromolar AA mimicsthrombin. Furthermore, inhibitors of 12-LOX, especially CDC, neutralizethe negative-chemotactic effect of thrombin. This supports the causalinvolvement of cPLA₂ because eicosanoid synthesis depends on thecellular supply of AA.

Role of Eicosanoids in Thrombin Signaling. The thrombin-like effect ofAA on pseudopod length could be due to its direct interaction with adownstream effector or it could occur indirectly, via a metabolite. Thecyclo-oxygenase inhibitor, indomethacin, did not interfere withAA-induced pseudopod shortening. In contrast the LOX blockers NDGA andCDC inhibited the AA effect completely. At the concentrations used, CDCis quite selective for 12-LOX suggesting that 12(S)-HETE is necessaryfor pseudopod shortening. This is supported by the fact that CDC blocksthe effects of thrombin.

While the 12-LOX inhibitor, CDC, inhibits the repellent effect ofthrombin, the product of 12-LOX, 12(S)-HETE, replicates the thrombineffect at very low concentrations (10⁻¹¹ to 10 M). Thus, the MAT-Luresponse to this eicosanoid is similar to the 12(S)-HETE-elicitedretraction of endothelial cells described in Tang ct al., Exp Cell Res207:361-375 (1993). Another eicosanoid, 15(S)-HETE, also causes MAT-Lupseudopods to withdraw, but the biologically inactive 12(R)-HETE, andanother isomer, 5(S)-HETE, cause only minimal or no changes in pseudopodlength. These data indicate potent, stereo-specific action of 12(S)-HETEand 15(S)-HETE on the MAT-Lu pseudopods.

The data indicate that thrombin acts on certain cell types, such as theMAT-Lu cells, or their pseudopods in a manner comparable to that ofrepellents on nerve growth cones. In more general terms, this suggeststhe operation of negative chemotactic mechanisms in non-neuralvertebrate tissues.

Our observations reveal the signaling mechanism involved in thrombin'srepellent action. The data indicate that cPLA₂ and 12-LOX are necessary,and that 12(S)-HETE is sufficient for the repellent effect. Furthermore,12(S)-HETE stimulates PKC, without the involvement of PLC, and PKCactivation is necessary for pseudopod detachment and withdrawal.

These examples establish (i) that certain growth cone repellentssuppress CaP cell migration, (ii) that such repellents trigger pseudopodadhesion site disassembly in CaP cells, and (iii) that this is mediatedby a signaling pathway involving cPLA₂, eicosanoid production and PKCactivation.

EXAMPLE 8 Thrombin-Induced Growth Cone Collapse Studies

The following studies were conducted to show that (a) PLA₂ activationand 12(S)-HETE generation are necessary for thrombin-induced growth conecollapse, (b) 12(S)-HETE mimics the thrombin response, and (c) asignalling pathway involving PLA₂ and 12-LOX regulates growth conedetachment.

1. Materials and Methods

Materials. Free arachidonic acid (AA) was purchased from Sigma (St.Louis, Mo.) and 1-stearoyl-2-arachidonyl-sn-glycerol (DG) fromAvanti-Polar Lipids (Alabaster, AL).L-α-stearoyl-2-¹⁴C-arachidonyl-phosphatidylinositol (¹⁴C-AA-PI; 20-40mCi/mmol), -phosphatidylethanolamine (¹⁴C-AA-PE; 40-60 mCi/mmol) and-phosphatidylcholine (¹⁴C-AA-PC; 40-60 mCi/mmol) were obtained fromDupont New England Nuclear (Boston, Mass.) as was ³H-arachidonic acid(³H-AA). Reagents for chemiluminescence were from the same vendor.Analytical grade solvents for routine thin-layer-chromatography (TLC)analysis and tissue culture dishes were purchased from Fisher(Pittsburg, Pa.). BDM (2,3-butanedione monoxime), TES(N-tris-[hydroxymethyl]-methyl-2-aminoethane-sulfonic acid), thrombinand other chemicals were from Sigma (St. Louis, Mo.). Aprotinin was fromCalbiochem (San Diego, Calif.). Polyacrylamide, pre-stained molecularweight standards and other reagents for sodium-dodecyl-sulphate(SDS)-polyacrylamide gel electrophoresis (PAGE) were from Gibco BRL(Grand-Island, N.Y.). Tissue culture media and laminin were also fromGibco BRL. HETE, nordihydroguaiaretic acid (NDGA),cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and indomethacin werepurchased from Biomol (Plymouth Meeting, Pa.). Thrombin receptoractivating hexapeptide TRAP) came from Peninsula Laboratories (SanCarlos, Calif.). TLC plates were obtained from EM Science (Gibbstown,N.J.) and from WHATMAN (Clifton, N.J.). Sprague-Dawley rats were fromHarlan (Indianapolis, Ind.). SPE columns were purchased from J. T. Baker(Phillipsburg, N.J.). Antibody to platelet-Lox was from OXFORDBiomedical Research (Oxford, Mich.) and antibody to leukocyte-Lox waspurchased from Cayman Chemical Company (Ann Arbor, Mich.).HRP-conjugated goat anti-rabbit antibody was purchased from VectorLaboratories (Burlingame, Calif.). Coverglasses and slides werepurchased from Carolina Biological Supply (Burlington, N.C.).Texas-Red-conjugated phalloidin and Slow-Fade Light were products ofMolecular Probes (Eugene, Ore.). Insulin and IGF-1 were fromCollaborative Biomedical Products (Bedford, Mass.) and BDNF from AmgenInc. (Thousand Oaks, Calif.).

Neuron Culture. Cerebral cortices were dissected from embryonic day 18Sprague-Dawley rats, cut into explants of less than 1 mm³ and culturedon either poly-D-lysine-coated coverglass (Assistent) orpoly-D-lvsine-coated tissue culture plastic. After 24 hours in B27neurobasal medium (NB) supplemented with 10% fetal bovine serum,cultures were switched to serum-free B27/NB. The cultures were incubatedat 37° C. in 5% CO₂ in air. Sprouting neurons were observed readily onday 1. On day 2 or 3, long neurites were present, and the cultures wereused for collapse experiments.

Actin Staining. After experimentation, cultures were fixed by slowinfusion of fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH7.4) into the culture dishes over 10 min (Pfenninger andMaylié-Pfenninger, J. Cell Biol. 89:53-546 (1981)). Thereafter, thefixative was removed by rinsing three times with phosphate-bufferedsaline (PBS) containing 1 mM glycine. Cultures were then blocked withPBS/1% bovine serum albumin (BSA) and permeabilized by incubation in0.02% Triton X-100 for 5 min. After three washes in PBS/BSA, cultureswere incubated with Texas-Red-conjugated phalloidin for 30 min. Unboundphalloidin was washed away by two PBS rinses, after which thecoverglasses were mounted in Slow-Fade Light on microscope slides.Images were recorded on 35 mm film using a Zeiss Axiophot fluorescencemicroscope equipped with epiillumination.

Growth Cone Collapse Assay. Prior to repellent factor treatment, some ofthe day 2-3 explant cultures were pre-incubated (at 37° C.) withinhibitor or vehicle control (dimethylsulfoxide or ethanol) for 30-60min. Cultures were then challenged with a repellent factor or vehiclecontrol for 1-30 min. In some experiments, live cultures werephotographed with a Zeiss IM 35 inverted photo microscope equipped witha stage heater, while in others cultures were fixed (as above) after 7minutes of repellent factor treatment. Growth cone collapse was assessedquantitatively in aldehyde-fixed cultures by grading the degree ofcollapse in a large number of randomly selected growth cones. Growthcones were classified in one of four categories: fully spread, “veiled”growth cones; partially spread growth cones possessing filopodia;partially collapsed growth cones with filopodia; and fully collapsed,“club”-shaped growth cones.

Growth Cone Particle (GCP) Isolation. GCPs were prepared as described byPfenninger et al. (1983) and modified by Lohse et al., Dev. Brain Res.,96:38-96 (1996). Briefly, 18-day fetal brains were homogenized inapproximately 8 volumes of 0.32 M sucrose containing 1 mM MgCl₂, 2 mMTES buffer, pH 7.3, and 3 μM aprotinin. A low-speed supernatant (1,660g×15 min) was loaded onto a discontinuous sucrose density gradient Withsteps of 0.83 M and 2.66 M sucrose containing MgCl₂ and TES. Thegradients were spun to equilibrium at 242,000 g for 40 min in a verticalrotor (Vti50, Beckman). The GCP fraction at the 0.32/0.83 M sucroseinterface was collected and used for the experiments described below.

GCP Release Assay. Petri dishes (35 mm diameter) were coated with 10 μgmouse laminin in PBS by incubation at room temperature for 60 min withshaking. Dishes were subsequently rinsed three times with PBS to removeunbound laminin and then blocked with 5% non-fat dry milk (Carnation) inPBS for 30 min. After three PBS rinses to remove blocking agent, thedishes were ready for the addition of GCPs. Prior to plating, the GCPfraction was slowly added at 4° C. to an equivalent volume of 2×concentrated, modified Krebs buffer (22 mM sucrose, 50 mM NaCl, 5 mMKCl, 22 mM Hepes pH 7.4, 10 mM glucose, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂ and2 mM CaCl₂). After incubation for five min at 37° C. this buffered GCPpreparation was added to the laminincoated petri dishes. Contact withthe substratum was facilitated by centrifuging the dishes for 15 min at5000 g (Beckman JS5.2 rotor) at room temperature, followed by incubationat 37° C. to allow adhesion site formation. After 10 min, unattachedGCPs were removed by rinsing the dishes twice with 1× modified Krebsbuffer. Plated GCPs were then challenged with repellent factor (TRAP orthrombin) for 20 min at 37° C. The supernatant containing released GCPswas collected and centrifuged for protein determination of the pellet.GCPs that remained attached after repellent treatment were removed with5% sodium dodecyl sulfate (SDS) for protein assay. Protein was measuredaccording to Lowry et al. (1951) as modified by Peterson (1983). Resultsof these experiments were expressed as the percentage of released versustotal GCP protein in the assay.

Gel Electrophoresis and Western Blotting. SDS-PAGE was performedaccording to Laemmli (1970), using 5-15% acrylamide gradients. The GCPfraction was diluted 3-4 fold with 0.32 M sucrose buffer and spun for 30min at 39,800 g. Protein amounts in the pellets were determined bydye-binding assay as described in Brafford, Anal Biochem 72:248-257(1976). Polypeptides of pelleted GCPs (30 μg/lane) were resolved bySDS-PAGE. Pre-stained standards were used to determine apparentmolecular mass. Resolved proteins were transferred to nitrocelluloseessentially as described by Towbin et al., Proc. Natl Acad Sci USA,76:4350-4354 (1979), with a semidry blotting apparatus for 60 min at 300mA for a 14×17 cm gel. Ponceau S staining served to monitor theefficiency of protein transfer. Blots were then rinsed in PBS anddistilled water and dried. Prior to incubation with primary antibody,blots were blocked with 5% non-fat milk powder and 0.02% Tween-20 in PBSfor two hours at room temperature. Blots were probed in the sameblocking solution and conditions with either polyclonal rabbitanti-platelet or polyclonal rabbit anti-leukocyte 12-Lox antibody at1:2000 or 1:5000, respectively. Blots were washed five times in blockingsolution, followed by incubation with secondary antibody (HRP-conjugatedgoat anti-rabbit antibody at 1:5000) for one hour in blocking solution.After extensive washing, bound antibody was detected by enhancedchemiluminescence according to the manufacturer's directions (NewEngland Nuclear, Boston Mass.) by contact-exposing x-ray film (KodakX-OMAT BLUE XB-1).

Phospholipase Assays. For PLA₂ assays, GCPs suspended in modifiedCa²⁺-free Krebs buffer (10-30 μg protein/assay) were first incubated for10 min on ice in the presence or absence of effectors. Subsequently,phospholipid substrate (PC, PE or PI) containing radiolabelled AA (7 μM;50,000 cpm/assay) was added and the reaction carried out at 37° C. for10 minutes. Typically, assay mixtures contained 10 μM CaCl₂; specificmodifications are described in the figure legends. For phospholipase C(PLC) assays, conditions were the same when run in parallel with PLA₂assays. When PLC assays were run independently, Ca²⁺ was increased to 1mM. Reactions were terminated by the addition of 3.75 volumes of coldchloroform/methanol (1:2). PLA₂ assays also were performed on neuronsdissociated from fetal rat cerebral cortex. The assay conditions werethe same as those just described for GCPs.

Extraction of phospholipase products was carried out according to Blighand Dyer, Canadien J Biochem and Physiol, 37:911-917 (1959). Productsrecovered in chloroform were loaded onto silica gel 60 TLC plates anddeveloped as described earlier (Nègre-Aminou and Pfenninger, 1993) inhexane/ether/acetic acid (40:60:1). AA and DG were identified on TLC byco-migration with authentic standards. The appropriate bands wereexcised into scintillation fluid and counted on a Beckman LS 1801scintillation sprectrometer. All assays were run in triplicate.

Eicosanoid Assays. Eicosanoid production in GCPs was measured bymetabolic labelling from ¹⁴C-AA or ¹⁴C-AA-PC or by mass spectrometricdetection. To determine HETE synthesis by radiolabelling, assays wererun essentially as for PLA₂ except that phospholipid substrate wasincreased to 100,000 cpm/assay, and assay time at 37° C. was 15 minutes.To measure Lox activity directly, without PLA₂ involvement, ³H-AA wasthe substrate of choice. Reactions were stopped by the addition ofchloroform/methanol as for the phospholipase assays. Reaction productswere extracted as described by Birkle et al., Neuromethods, vol. 7,pp.227-244 (Humana Press, N.J. 1988), spotted on pre-activated si gel 60TLC plates, and the plates developed in the upper phase of ethylacetate/isooctane/acetic acid/water (100:60:20:100). AA and HETEs wereidentified by co-migration with standards. Appropriate bands werecounted by liquid scintillation spectrometry.

For mass-spectrometric (MS) identification of HETEs, two differentstrategies were pursued:

(1) the appropriate bands from non-labelled samples (PC substrate) werescraped from the TLC plate into ether/methanol (9:1) and stored under N₂at −20° C. Authentic 12(S)-HETE, run alongside in the same manner, wasused as a standard. Just prior to analysis, samples were passed over anSPE filter to eliminate silica particles, dried under argon, resuspendedin 500 μl ethanol, and injected (at 3 μl/min) into a Finnigan LCQquadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, Calif.)equipped with an electrospray ionization source. All spectra wereacquired at a capillary temperature of 80° C., and ion guide voltageswere tuned to maximize the abundance of the deprotonated ion species[M-H]⁻ in negative mode. In order to generate a spectrum, typically 100single scans were averaged.

(2) For the other strategy (liquid chromatography/tandem MS, LC/MS²),the reaction substrate consisted of 3.5 μM AA, with the addition of 3.5μM deuterated AA (AA-D₈) plus 1 μCi ³H-AA to track reaction products bymass and radioactivity, respectively. Reactions were stopped withice-cold ethanol, the mixtures kept overnight at −20° C. under N₂, andthe proteins spun out The supernatants were diluted with H₂O and loadedonto 1-ml C₁₈ Sep-Pak columns (Varian). After washing with H₂O,eicosanoid was eluted with 2 ml methanol. These samples were resolved byreverse-phase HPLC, and the eluted fractions analyzed by tandem MSon-line (MacMillan and Murphy, 1995). Collision-induced decomposition ofHETE isomers produced characteristic fragments that were identified atthe second MS stage.

2. Results

Growth Cone Collapse in Culture. Experiments were performed on growthcones in cultures of rat cerebral cortex to assess qualitatively andquantitatively the collapsing effects of thrombin and thenon-proteolytic TRAP, with or without inhibitors of AA metabolism. Inthese studies, cultures of E18 rat cortical neurons were eitherpretreated with vehicle alone or inhibitor for 45 minutes prior tocollapse factor treatment with thrombin or 12(S)-HETE. The treatmentsincluded: (1) 100 nM thrombin treatment; (2) 25 μM indomethacinpretreatment followed by 100 nM thrombin; (3) 10 μM CDC pretreatmentfollowed by 100 nM thrombin; (4) 10⁻⁷ M 12(S)-HETE treatment of growthcones at different times. Within minutes, thrombin causes disappearanceof lamellipodia and, eventually, filopodia. After 10-20 min, most growthcones are fully collapsed and neurites exhibit beading and lengthreduction. TRAP was used at a concentration about 1000 times higher thanthat of thrombin, consistent with data on receptor binding and TRAPactivation in other systems. TRAP has qualitatively the same effect asthrombin, but the change is not as pronounced, and neurite beading isnot as common. The collapse phenomenon can be seen at highermagnification after staining of filamentous actin withTexas-Red-conjugated phalloidin. Control growth cones exhibit spread-outveils and/or filopodia with filamentous actin enriched in the peripherywhereas thrombin or TRAP treatment causes withdrawal of lamellipodia andfilopodia and actin redistribution from the peripheral to the proximalgrowth cone.

In order to test for the putative role of PLA₂ in thrombin/TRAPsignalling, it would ideally inhibit or activate growth cone PLA₂.However, the molecular identities of growth cone PLA₂s have not beenestablished, and selective inhibitors that block these enzymes are notknown. Therefore, we proceeded with experiments involving inhibitors ofthe metabolism of the PLA₂ product, AA. Indomethacin is a specificblocker of cyclooxygenase, thereby inhibiting the synthesis ofprostaglandins and related eicosanoids (Salari et al., ProstaglandinsLuckotrienes and Medicine, 13:53-60 (1984)). As indicated above, CDC isa selective inhibitor of Loxes, including 12-Lox, which catalyzes thesynthesis of 12(S)- and some 15(S)-HETE. Indomethacin has no effect oncontrol or thrombin treatment of growth cones. However, pretreatmentwith CDC inhibits the thrombin effect on growth cones. While thrombincollapses growth cones even after incubation with indomethacin,CDC-pretreated growth cones retain their spread-out appearance. A morepotent but less selective inhibitor of all Loxes, NDGA (Salari et al.,1984), also protects growth cones from thrombin- or TRAP-inducedcollapse.

Comparisons were done of phalloidin-stained growth cones, in controlcultures and in cultures challenged with thrombin or TRAP after CDCincubation. Cultured E18 rat cortical neurons were fixed and stainedwith Texas-Red-conjugated phalloidin after the following experimentaltreatments: control (vehicle alone); thrombin (100 nM) for 7 minutes;TRAP (100 mM) for 7 minutes; 45 minutes pretreatment with CDC (10 μM)followed by TRAP (100 mM) for 7 minutes; 12(S)-HETE (10⁻⁷ M) for 10minutes. Thrombin and TRAP cause collapse but the TRAP effect, whileclear-cut, is not as dramatic as that of thrombin (arrows point atcollapsed growth cones); CDC partially inhibits collapse; and 12(S)-HETEmimics the effects of thrombin and TRAP. The Lox inhibitor, CDC,inhibits thrombin-induced shape change of the growth cone, but notretraction of the actin cytoskeleton. Growth cones were fixed andstained with Texas-Red-conjugated phalloidin after control (vehiclealone) incubation and pretreated 30 minutes with CDC (1 μM), followed bythrombin challenge (100 nM) for 7 minutes. Intact filopodia withdiminished actin staining were seen as well as “clumped” actin filamentsin the proximal growth cone. This illustrates an important point: eventhough CDC-pretreated growth cones typically retain their spread-outshape with attached filopodia, a substantial amount of filamentous actinappears clumped in the proximal body of many of the growth cones. Someactin de-polymerization may have taken place as well. This observationsuggests a dissociation of cytoskeletal redistribution (not inhibited)from filopodial detachment (inhibited by CDC).

The results of these experiments have been analyzed quantitatively. Atleast 50 growth cones in each of 4 to 6 experiments were classified intofour different categories: collapsed, partially collapsed, partiallyintact, and intact (see Methods). FIG. 17 shows a shift of growth conesfrom predominantly intact (controls) to predominantly collapsed(thrombin or TRAP) and the almost complete inhibition of this effect byCDC.

If 12-Lox is necessary for at least a part of the collapsing action ofthrombin/TRAP, as the experiments with CDC suggest, then the primary12-Lox product, 12(S)-HETE, should mimic the effect of thrombin/TRAP.This is shown quantitatively in FIG. 17. In this system at 10⁻⁷M,12(S)-HETE does indeed cause growth cone collapse that ismorphologically very similar to the effect of thrombin/TRAP. However,12(S)-HETE-induced collapse is less dramatic (see FIG. 17), and theneurite beading seen after maximum thrombin exposure is observed onlyrarely. The collapse effect of 12(S)-HETE is not affected by growth conepretreatment with the Lox inhibitor, CDC.

Growth Cone Detachment. Morphologic analysis of growth cones, asdescribed above, suggests that cytoskeletal redistribution andfilopodial detachment during collapse are separate phenomena. In orderto test this hypothesis further we designed a cell-free substratedetachment assay involving so-called growth cone particles (GCPs)isolated from fetal rat brain (Pfenninger et al., 1983). GCPs contain afull complement of growth cone organelles, are viable for at least onehour after isolation and have been shown to be derived primarily fromaxonal growth cones (Lohse et al., 1996). When GCPs attached to lamininwere exposed to thrombin, many of them detached from the substratum in adose-dependent maaner and could be collected in the supernatant (FIG.18). TRAP mimics the effect, indicating that protease activity is notrequired for detachment.

GCPs contain a substantial actin meshwork (Pfenninger et al., 1983).Despite their small size (about 0.3-0.5 μm diameter), contraction of theactin/myosin system could be responsible for at least some of thedetachment effect observed in the assay. Therefore, laminin-attachedGCPs were first treated for 20 min at 37° C. with 1 μM cytochalasin D todepolymerize the actin cytoskeleton or with 20 mN BDM to inhibitselectively myosin ATPasc. Both reagents caused considerable release ofGCPs by themselves (FIG. 18) consistent with known effects on cellspreading. However, thrombin challenge increased detachment further, by1.5 to 1.75 fold (p<0.05 for BDM; p<0.01 for cytochalasin D).

Phospholipase Activation. Our hypothesis predicts that one of theimportant steps in thrombin-induced growth cone collapse is theactivation of cytosolic PLA₂. Therefore, we studied PLA₂ activation inprimary neurons and in GCPs isolated from fetal rat brain. In PLA₂assays performed on primary neurons dissociated from cerebral cortex,thrombin greatly stimulated AA release from PE or PI, as shown in Table3a. Because of the specific effect of thrombin on growth cones andbecause of the enrichment of PLA₂ in growth cones (Nègre-Aminou et al.,1996), we proceeded with the more detailed analysis of PLA₂ activationin GCPs. Table 3a shows that, under comparable experimental conditions,thrombin stimulates PLA₂ in GCPs to much higher levels of free AA thanin whole neurons. FIG. 6 shows the release of AA from PI as a functionof increasing concentrations of thrombin or TRAP. We observed a highlevel of activation (from 5- to 7-fold) saturating at about 250 nM forthrombin. TRAP also clearly stimulated PLA₂, but to a considerablylesser degree and at a roughly 1000-fold higher concentration. We haddetermined previously that GCPs contain very little, if any, secretedPLA₂ (Nègre-Aminou et al., 1996). In order to exclude the possibilitythat thrombin stimulated the release of a secreted PLA₂ we performedPLA₂ assays in the presence of reducing agent, which inactivates thecatalytic domain of all secreted forms of the enzyme. As Table 3b shows,dithiothreitol (DTT) does not reduce the control or thrombin-stimulatedlevels of PLA₂ activity. This confirms the role of cytosolic PLA₂ in athrombin-activated pathway.

The PLA₂ response to thrombin is calcium-dependent in permeabilizedGCPs, as shown in FIG. 20. In the presence of EGTA, there is nostimulation of enzyme activity, whereas the thrombin response reaches amaximum at about 100 μM Ca²⁺, without much change in basal,non-stimulated conditions.

In a previous publication (Nègre-Aminou et al., 1996), we reported thatGCPs contain at least two biochemically separable PLA₂ activitiesselective for PI and PE, respectively. Under experimental conditionsused at the time, PC hydrolysis in GCPs was at background level withoutstimulation. However, FIG. 21 shows that thrombin stimulates AA releasefrom all three phospholipid substrates, but at different levels, rangingfrom about 5-fold for PI to about 7-fold for PC.

In other systems, such as platelets, thrombin has been reported toactivate PLC (Crouch and Lapetina, J. Biol. Chem., 263:3363-3371 (1998);Huang et al., J. Biol. Chem., 266:18435-18438 (199)). Therefore, wemeasured diacylglycerol (DG) release from PI in parallel to AA release.FIG. 22 shows that thrombin inhibits DG release, rather than stimulatingit, and that the effect is calcium-dependent in a manner similar to thestimulation of PLA₂. In order to ascertain the authenticity ofthrombin-induced PLC inhibition, we compared the effects of thrombin andinsulin-like growth factor 1 (IGF-1) on PLA₂ and PLC in GCPs. GCPs areknown to be rich in IGF-1 receptors and IGF-1 is known to activate PLCin many systems. FIG. 23 shows AA and DG release from PI (at 10 μM freeCa²⁺) in response to different concentrations of IGF-1. In the lownanomolar range, IGF-1 does indeed stimulate DG release in GCPs,approximately two-fold (FIG. 23). IGF-1 also stimulates AA release fromPI, but only about 1.7 fold. This acuvanon or PLA₂ is a much weakerresponse than that observed with thrombin (see FIGS. 19 and 21).

Because of the mild stimulation of PLA2 by IGF-1, we assayed for PLA₂activation by other trophic factors whose receptors are known to bepresent on growth cones or to be linked to PLA₂ stimulation in othercellular systems. (In these experiments, shown in Table 3, net controllevels of PLA₂ activity in GCPs range from 38 to 107 pmol AAreleased/min/mg protein, probably because of slight variations in theGCP fraction. However, data sets are shown always with controls measuredin the same experimental series.) TrkB, the receptor for brain-derivedneurotrophic factor (BDNF), is readily detectable by immunoblot andenriched in GCPs isolated from whole fetal brain. However, BDNF did notsignificantly stimulate PLA₂ activity in GCPs (Table 3c). BDNF combinedwith IGF-1 or insulin alone also failed to stimulate PLA₂ significantlyin our assays (Table 3c).

TABLE 3 PLA₂ Activity in Neurons and Growth Cones a) Whole neuronsversus GCPs. net activity (pmol/min/mg protein) condition concentrationnM substrate intact neurons GCPs control — PI  0.6 ± 0.45  37.9 ± 1.1 PE 4.4 ± 0.99  25.7 ± 2.3 thrombin 100 PI 42.5 ± 16     262 ± 47 PE 26.8 ±1.9 210.2 ± 4.8 b) Effect of reduction on PLA₂ activity in GCPs. netactivity (pmol/min/mg protein) condition concentration nM substrate noDTT 50 nM DTT control — PI 41.9 ± 1.1  66.0 ± 4.7  thrombin 100 PI  221± 39.9  223 ± 53.4 c) Response of GCPs to different factors. netactivity condition concentration nM substrate (pmol/min/mg protein)control — PI 159 ± 6.2  BDNF 100 PI 164 ± 10.6 IGF-1 0.75 PI 177 ± 14.5BDNF + IGF-1 100/0.75 PI 154 ± 12.4 insulin 1 PI 158 ± 8.4  thrombin 200PI 355 ± 28.0

Generation of Eicosanoid. The effect of CDC in collapse and growth conedetachment assays suggests that eicosanoid synthesis from AA is animportant step in thrombin/TRAP signalling. Lipid extracts prepared inacid conditions can be resolved by TLC to separate 12- and 15-HETE(which co-migrate) from the other compounds, including AA, DG and 5-HETE(Birkle et al., 1988). With this approach we studied the generation ofHETE in GCPs, without or with thrombin stimulation. FIG. 24A shows, inassays involving ¹⁴C-AA-PC as a substrate, the generation of AA and of acompound co-migrating in TLC with 12- or 15-HETE. As seen already, thereis substantial stimulation of AA release by thrombin. Radioactivityco-migrating with 12-HETE also is increased, approximately 2.5-foldabove control (FIG. 24B). The selective 12-Lox inhibitor, CDC, reducesthrombin-stimulated HETE levels to below control, suggesting that thiscompound is indeed 12- and/or 15-HETE. This experiment indicates thatthrombin stimulates HETE synthesis in GCPs, but it does not discriminatebetween the activation of PLA₂ (shown by increased AA release) and thepossible stimulation of Lox activity.

In order to determine whether thrombin regulates Lox activity, analogousassays were performed with ³H-AA as the substrate (total AAconcentration, about 8 μM. As shown in FIG. 24C, thrombin does indeedstimulate HETE production from AA, and the effect is strongly inhibitedby CDC, but not by the cyclooxygenase blocker, indomethacin. Thissuggests thrombin activation of Lox, in addition to PLA₂. However, theamount of HETE generated upon thrombin stimulation is only a smallfraction of the amount of AA released (FIG. 24A).

Although TLC is suitable for isolating eicosanoids, identification ofthe compounds is based on co-migration with standards. Therefore, weperformed mass-spectrometric analysis (a) to ascertain HETE identity ofthe ¹⁴C-AA-PC- or ³H-AA-derived substance co-migrating in TLC with12(S)-HETE and (b) to identify HETE isomers endogenously generated bygrowth cones. HETE isomers have a negative ion m/z of 319. However, when12(S)-HETE is first run on TLC, subsequently desorbed from the silicaand then analyzed by MS, it generates a primary negative ion peak at m/z381. Extracts from control GCPs subjected to the same procedure alsocontain a predominant peak at m/z 381, in the TLC band co-migrating with12(S)-HETE. This peak is increased nearly 2-fold if GCPs are firststimulated with thrombin. Thus, MS supported the assumption that the TLCband analyzed to determine Lox activity contained 12- or 15-HETE.

LC/MS² was used to demonstrate GCP synthesis of specific HETE isomer(s).FIG. 25 shows HPLC elution patterns. The abscissa indicates relativedetection levels of fragments derived from HETE isomers (negative ionm/z 319.3) and characteristic of 12-HETE (fragment m/z 179.1) and15-HETE (fragment m/z 219.1), respectively. Based on scale and peakwidth, 12-HETE is the predominant species, but a significant level of15-HETE is evident. 5-HETE was not detected.

The generation of 12- and 15-HETE by growth cones, the inhibition ofthis synthesis by CDC (at 0.156 μM), and the inhibition of the thrombineffect on growth cone morphology by CDC suggest that growth conescontain one or several forms of 12-Lox. To test for this possibility, weanalyzed GCPs by Western blot with the two 12-Lox antibodies currentlyavailable, specific for platelet and leukocyte 12-Lox, respectively.Gels contained equal amounts of protein from fetal-brain low-speedsupernatant (LSS, the crude parent fraction of GCPs) and GCPs. Theresults indicate that GCPs are significantly enriched, relative to LSS,in a protein of about 75 kDa and reactive with the antibody to leukocyte12-Lox. No immunoreactivity was detected with anti-platelet 12-Loxantibody.

3. Discussion

Thrombin as a Growth Cone Repellent. Thrombin receptor activation leadsto diverse cellular responses, such as secretion and pseudopod spreadingin platelets, and locomotion and mitogenesis in fibroblasts. However, itcauses cell rounding in endothelial and neural cells and pseudopodwithdrawal in certain cancer cells (Grand et al., Biochem J. 313:353-368(1996); Jalink and Moolenaar, J. Cell Biol., 118:411-419(1992);Vouret-Craviari et al., Mol. Biol. Cell, 9:2639-2653 (1998)). Inneurons, thrombin induces growth cone collapse comprising redistributionand partial depolymerization of the actin skeleton as well as detachmentof filopodia and lamellipodia. This is very similar to collapse inducedby other factors (e.g., Kapfhammer and Raper, J. Neurosci,7(5):1595-1600 (1987; Fan et al., J. Cell Biol., 121:867-878 (1993)).TRAP mimics the effect of thrombin, indicating that this is areceptor-mediated event, not dependent on proteolysis of adhesionmolecules or their extracellular ligands. Thus, thrombin qualifies as abona-fide collapsing factor and repellent.

Activation of Phospholipase and Lipoxygenase. In plaelets, thrombinactivates both cPLA₂ and PLC. It seemed logical, therefore, to assay forthese enzymes in cultures of thrombin-responsive primary neurons.Isolated growth cones were of particular interest in view of theenrichment of at least two different cPLA₂s in them (Nègre-Aminou etal., 1996; whether the two enzymes occur separately in distinct growthcone populations, or together in all GCPs in unknown).

Thrombin and, to a lesser extent, TRAP stimulate PLA₂ activity inprimary neurons and GCPs, with the highest levels of free AA achieved inGCPs. Growth factors whose receptors are present on GCPs, such as BDNF,NGF, insulin and IGF-1, stimulate growth cone cPLA₂ only weakly or notat all, indicating that thrombin activation is selective. Thrombinincreases AA release from phospholipid from 7- to 5-fold, with thesubstrate preference PC>PE>PI. Resting as well as stimulated levels ofPLA₂ activity are resistant to reducing agents, excluding a contributionof secreted PLA₂. PC hydrolysis had not previously been detected inunstimulated GCPs (Nègre-Aminou et al., 1996). Thus, present resultssuggest a third, PC-selective cPLA₂ may possibly exist in GCPs. We showthat thrombin activation of cPLA₂ requires Ca²⁺. However, Nègre-Aminouet al. (1996) reported PE- and PI-selective cPLA₂s to beCa²⁺-independent. Therefore, Ca²⁺ is likely to be necessary for asignalling step upstream of cPLA₂.

While growth cones and platelets share receptor-mediated thrombinstimulation of cPLA₂, they are different with regard to PLC regulation.Thrombin stimulates platelet PLC but inhibits this phosphoinositidase inGCPs. PLC activation causes the release of IP₃ and subsequent efflux ofCa²⁺ from intracellular storage sites. Our observations thus suggestthat release of Ca²⁺ from intracellular stores is not important for thecollapse response. Thrombin-induced Ca²⁺ transients have been reportedfor platelets (see Grand et al., 1996) and neuroblastoma cells (Jalinkand Moolenaar, 1992), but they were shown not to be necessary forthrombin-induced rounding of neuroblastoma and endothelial cells (Jalinkand Moolenaar, 1992; Vouret-Craviari et al., 1998). Therefore, ourfindings are consistent with these observations as well as the fact that(at least so far) Ca²⁺ transients have not been detectable in nervegrowth cones treated with repellents. The second consequence of PLCstimulation, the release of DG, activates PKC in platelets. Lack of PLCstimulation in thrombin-treated GCPs, however, does not rule out a rolefor PKC in repellent action.

cPLA₂s cleave phospholipids into an unsaturated fatty acid, in braintypically AA, and a lysophospholipid. Whether lyso-PI, -PE and -PCgenerated in growth cones have functional role(s) is not known. Mostreleased AA is rapidly reincorporated into phospholipid (Nègre-Aminouand Pfenninger, 1993), but AA may directly influence growth conefunctions and/or may be converted into one or more eicosanoids (Smith,Biochem J., 259:315-324 (1989); Shimizu and Wolfe, J. Neurochem.,55:1-15 (1990)). Our biochemical studies show that thrombin stimulatesin growth cones not only the release of AA but also the synthesis of anAA-derived compound that co-extracts and co-migrates in TLC with 12- or15-HETE. The generation of this product is inhibited by the selective12-Lox blocker, CDC, but not by the cyclooxygenase inhibitor,indomethacin. MS analysis of the material eluted from TLC at the sameR_(f) reveals a single major peak at m/z 381, the negative ion m/z seenfor pure 12(S)-HETE subjected to the same extraction and TLC protocol.(The observed compound may be an acetylated and reduced derivativeproduced during TLC, as suggested by variations of the protocol). Inagreement with the radiolabel data, the peak seen at m/z 381 isincreased significantly by thrombin stimulation of GCPs prior toextraction. Finally, LC/MS² analysis of GCP extracts definitivelydemonstrates GCP synthesis of primarily 12- and also 15-HETE, but not of5-HETE.

In summary, our results demonstrate strong and selective thrombinstimulation of one or multiple cPLA₂s in growth cones, followed byconversion of some of the released AA into 12- and 15-HETE. We estimatethe conversion to amount to about 1 pmole, or 5%, out of approximately20 pmoles AA released in the same assay (assuming equal recoveries).Although we cannot exclude a role of 15-Lox in GCPs, HETEs are likely tobe synthesized by 12-Lox because of the selective inhibitory effect ofCDC at 0.156 μm (Cho et al., 1991) and a recent report that 12-Lox alsogenerates 15 (Yamamoto, Prog. Lipid Res. 36(1):23-41 (1991)). This viewis consistent with the observation that growth cones are enriched in apolypeptide that co-migrates in SDS-PAGE with 12-Lox (at 75 kDa) andimmuno-crossreacts with an antibody to leukocyte-type 12-Lox (but notplatelet 12-Lox).

It is generally assumed that eicosanoid synthesis, including that of12-HETE, is regulated by the supply of AA, and that 12-Lox isconstitutively active (Smith, 1989; Shimizu and Wolfe, 1990; Yamamoto etal., 1997). However, we observed that thrombin stimulates not only cPLA₂but also the Lox in GCPs.

Functional Role of Lipid Messengers in Growth Cone Collapse. The datadiscussed so far correlate thrombin's collapsing effect with cPLA₂activation and eicosanoid synthesis. In order to determine whether acausal relationship exists, inhibitor experiments were performed.Selective inhibitors of most or all growth cone cPLA₂ activity are notknown, but exist for eicosanoid synthesis. The cyclooxygenase inhibitorindomethacin does not interfere with thrombin-induced growth conecollapse. However, the general Lox inhibitor, NDGA, and the specific12-Lox inhibitor, CDC, block thrombin- and TRAP-induced collapse, aswell as Lox activity in biochemical assays. Conversely, exogenous12(S)-HETE added to cultures mimics the effects of thrombin or TRAP.These results are consistent with the observed thrombin stimulation ofHETE synthesis and link cPLA₂ activation and HETE synthesis functionallyto the collapse mechanism.

As pointed out earlier, growth cone collapse involves filopodial andlarmellipodial detachment and withdrawal, as well as reorganization ofthe actin cytoskeleton. Our growth cone detachment assay and the effectof CDC dissociate these two phenomena: Thrombin and TRAP trigger GCPdetachment from laminin. Disassembly of the actin cytoskeleton withcytochalasin D or inhibition of myosin ATPase with BDM interfere to somedegree with GCP attachment, but rather than blocking thrombin-induceddetachment, they facilitate it. So, detachment does not require anintact actin cytoskeleton, at least in these assays. A complementaryobservation is that intact growth cones pretreated with CDC and thenchallenged with thrombin retain their spread-out, attached filopodia andoverall shape, but phalloidin staining often reveals peripherallydepleted and proximally condensed F-actin. These results taken togethersuggest that cPLA₂ and 12-Lox are involved in triggering detachmentrather than cytoskeletal redistribution. Because Lox inhibitors do notseem to affect thrombin-induced change of the actin cytoskeleton,signalling to this entity seems to follow an alternate pathway,presumably involving Rho-family G-proteins (see Jalink et al., 1994;Vouret-Craviari, 1998).

In conclusion, our data suggest that the growth cone repellent,thrombin, activates a branching signalling cascade, with one pathleading to redistribution of the actin cytoskeleton and the other(involving cPLA₂ and 12-Lox) triggering pseudopod detachment. Theresults indicate that eicosanoid synthesis is necessary and the 12-Loxproduct, 12(S)-HETE, sufficient to cause pseudopod detachment,presumably via disassembly of adhesion sites. The modes of action of12(S)- and 15(S)-HETE are not known, but our preliminary data suggestthe involvement of PKC activation and phosphorylation of the adhesionsite-associated protein, MARCKS and/or MacMARCKS (de la Houssaye et al.,Mol. Biol. Cell, 8:265a(1997); Mikule et al., Soc. Neurosci Abstracts,23(part 1), p. 600 (1997)).

While the exemplary preferred embodiments of the present invention aredescribed herein with particularity, those having ordinary skill in theart will recognize various changes, modificatins, additions, andapplications other than those specifically described herein, and mayadapt the preferred embodiments and methods without departing from thespirit of the invention.

What is claimed is:
 1. A method of identifying an agent that reduces theability of a neurite repellent to inhibit neurite growth, comprising: a.attaching neuronal growth cones to a substratum; b. exposing theattached neuronal growth cones to a putative agent in the presence of aknown repellent; and c. determining the amount of attached or detachedneuronal growth cones, wherein the putative agent reduces the ability ofa neurite repellent to inhibit neurite growth if more of the neuronalgrowth cones remain attached to the substratum in the presence of saidputative agent and said known repellent as compared to in the absence ofsaid putative agent and in the presence of said known repellent.
 2. Themethod of claim 1, wherein the neuronal growth cones are attached on asubstratum in a modified Kreb's buffer.
 3. The method of claim 1,wherein the step (a) of attaching said neuronal growth cones to asubstratum comprises centrifuging said neuronal growth cones on saidsubstratum for up to sixty minutes at a speed of at least about 2,000×g.4. The method of claim 1, wherein the step (a) of attaching saidneuronal growth cones to a substratum comprises centrifuging saidneuronal growth cones on said substratum for about 15 minutes at about5,000×g at room temperature.
 5. The method of claim 1, wherein step (c)is determined by the amount of neuronal growth cones that remainattached to the substratum, wherein the putative agent is effective toreduce the ability of a repellent to inhibit neurite growth if at least75% of the neuronal growth cones remain attached to the substratum. 6.The method of claim 1, wherein step (c) is determined by the amount ofneuronal growth cones that remain attached to the substratum, whereinthe putative agent is effective to reduce the ability of a repellent toinhibit neurite growth if at least 85% of the neuronal growth conesremain attached to the substratum.
 7. The method of claim 1, whereinstep (c) is determined by the amount of neuronal growth cones thatremain attached to the substratum, wherein the putative agent iseffective to reduce the ability of a repellent to inhibit neurite growthif at least 95% of the neuronal growth cones remain attached to thesubstratum.
 8. The method of claim 1, wherein the substratum comprisesextracellular matrix molecules or cellular adhesion molecules.
 9. Themethod of claim 8, wherein the extracellular matrix molecules arelaminin, fibronectin, collagen or a combination thereof.
 10. The methodof claim 8, wherein the cellular adhesion molecules are L1, N-CAM,cadherin, glycolipids, synthetic polypeptides, oligosaccharides or acombination thereof.
 11. The method of claim 1, wherein the substratumis coated with nitrocellulose.
 12. The method of claim 1, wherein theknown repellant is selected from the group consisting of thrombin and asemaphorin.
 13. The method of claim 1, wherein said method furthercomprises a step of measuring a parameter selected from the groupconsisting of neuronal growth cone elongation, neuronal growth coneretraction, cell extension, adhesion site formation, adhesion sitedetachment, and activation of a parameter within the signaling pathway.14. The method of claim 1, wherein said method further comprises a stepof measuring whether the putative agent inhibits the activation of aparameter in the repellent signaling pathway.
 15. The method of claim14, wherein said parameter in the repellent signaling pathway isselected from the group consisting of cytosolic phospholipase A₂(cPLA₂), 12-lipoxygenase (12-LOX) and protein kinase C.
 16. The methodof claim 1, wherein said known repellent agent is identified by a methodcomprising: a. attaching neuronal growth cones on a substratum; b.exposing the attached neuronal growth cones to a putative repellentagent; and c. determining the effect of the putative repellent agent onthe neuronal growth cones, wherein detachment of at least about 35% ofthe neuronal growth cones from the substratum in the presence of theputative repellent agent indicates the putative repellent agent is aneffective repellent agent.
 17. The method of claim 1, wherein said knownrepellent is identified by a method comprising: a. obtaining wholeneural cells or neuronal growth cones; b. exposing the whole neuralcells or neuronal growth cones to a putative repellent agent; and c.measuring a parameter on the repellent signaling pathway, wherein theputative repellent agent inhibits cell motility if the parameter isactivated.